Compositions and methods for promoting healthy neural development in an unborn baby

ABSTRACT

Disclosed herein are methods for promoting healthy neural development in an unborn baby, which include administering to a maternal subject gestating the unborn baby a composition or a bacterial composition. Compositions can include trimethylamine N-oxide (TMAO), 5-aminovalerate (5-AV), imidazole propionate (IP), hippurate (HIP), or a combination thereof, and the bacterial compositions can include bacteria of the order Clostridiales. Also disclosed are methods for conditioning a female subject for bringing about offspring with healthy neural development. Additionally disclosed are methods for reducing adverse effects of antibiotic treatment on an unborn baby in a pregnant subject. Also disclosed are methods for selecting a female subject for conditioning to foster healthy neural development in offspring.

RELATED APPLICATION

This application claims a right of priority to and the benefit of U.S. Provisional Application No. 62/844,503, filed on May 7, 2019, which is hereby incorporated by reference herein in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant Numbers AI007323, GM106996, and DE025172, awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND

Dysbiosis of the maternal gut microbiome, in response to environmental challenges such as infection, altered diet, and stress during pregnancy has been increasingly associated with abnormalities in offspring brain function and behavior. However, whether the maternal gut microbiome begins to exert its influences during critical periods of embryonic brain development remains poorly understood.

The intestinal microbiota is an important modulator of brain function and behavior, but further research is necessary to resolve whether there are prenatal critical periods during which the microbiome impacts the development of the nervous system. Various model organisms reared devoid of microbial colonization (germ-free, GF) or depleted of the gut microbiome (antibiotic-treated, ABX) exhibit altered neurophysiology and behavior compared to conventionally-colonized (specific pathogen-free, SPF) controls. Only a subset of phenotypes can be corrected by postnatal restoration of the microbiome, suggesting a role for the early life microbiome in regulating developmental processes that impact brain function and behavior during adulthood. Thus, methods of modifying the maternal microbiome, for example to compensate for a depleted maternal microbiome, prenatally (i.e., during gestation) are needed.

SUMMARY

In some aspects, methods of promoting healthy neural development in an unborn baby include administering to a maternal subject gestating the unborn baby a composition that comprises trimethylamine N-oxide (TMAO), 5-aminovalerate (5-AV), imidazole propionate (IP), hippurate (HIP), or a combination thereof.

In certain aspects, methods of reducing adverse effects of antibiotic treatment on an unborn baby in a pregnant subject include administering to the pregnant subject, conjointly with the antibiotic treatment, a composition comprising trimethylamine N-oxide (TMAO), 5-aminovalerate (5-AV), imidazole propionate (IP), hippurate (HIP), or a combination thereof.

In some aspects, methods of conditioning a female subject for fostering healthy neural development in offspring are disclosed. These methods include administering to the female subject a composition comprising trimethylamine N-oxide (TMAO), 5-aminovalerate (5-AV), imidazole propionate (IP), hippurate (HIP), or a combination thereof, e.g., in which the composition is administered at least once during a period that runs from the first day of an expected-but-missed menstruation to a day that is two months after that first day. In some embodiments of these aspects, the composition is administered at least once during a period that runs from the second day of the expected-but-missed menstruation to a day that is 10 to 60 days (e.g., 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 35, 36, 37, 38, 39, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60) after said second day.

These aspects have many embodiments. For example, in certain embodiments healthy neural development includes healthy tactile sensory development. In some embodiments, the composition includes 5-AV and IP. In certain embodiments, the composition includes TMAO. In certain embodiments, healthy neural development includes healthy thalamocortical axon growth. In some embodiments, healthy neural development includes healthy netrin-G1a+thalamocortical axogenesis. The maternal subject and the unborn baby are preferably mammals, most preferably primates, especially humans.

In particularly preferred embodiments, the maternal subject and the unborn baby are humans. In certain such embodiments, the method includes administering the composition at least once during the first trimester of the gestating maternal subject's gestation period. In some embodiments, the method includes administering the composition at least once during a period that runs from the start of the third week after conception to the end of the eighth week after conception. In certain embodiments, the method includes administering the composition at least once during a period that runs from the 17^(th) day post conception (dpc) to the 52^(nd) dpc. This period can be varied, for example, it can start from any of the following dpcs: 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 and end at any one of the following dpcs: 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 70, 80, 90, 100, 150, 200, 250. In some embodiments, the method includes administering the composition at least once during the second trimester of the gestating maternal subject's gestation period. In certain embodiments, the method comprises administering the composition at least once during the third trimester of the gestating maternal subject's gestation period. In some embodiments, the unborn baby is an offspring of the maternal subject.

In some aspects, methods of promoting healthy neural development in an unborn baby include administering to a maternal subject gestating the unborn baby a bacterial composition comprising bacteria of the order Clostridiales. In some such embodiments (e.g., of any aspect involving the use of, as well as other embodiments relating to the use of bacteria of the order Clostridiales herein), the bacteria of the order Clostridiales include bacteria of the family Lachnospiraceae, family Ruminococcaceae, family Clostridiaceae, or a combination thereof. In certain embodiments, the bacteria of the order Clostridiales include bacteria of the genus Clostridium, genus Dehalobacterium, genus Ruminococcus, genus Coprococcus, genus Dorea, genus Oscillospira, or a combination thereof. In some embodiments, the bacteria of the order Clostridiales are spore-forming bacteria.

In certain embodiments (e.g., of any aspect involving the use of bacteria of the order Clostridiales), the method includes administering the bacterial composition at least once during the first trimester of the gestating maternal subject's gestation period. In some embodiments, the method further includes administering the bacterial composition at least once during the two-month period before said gestation period starts. In certain embodiments, the method further includes administering to the maternal subject a composition comprising trimethylamine N-oxide (TMAO), 5-aminovalerate (5-AV), imidazole propionate (IP), hippurate (HIP), or a combination thereof.

In certain aspects, methods of conditioning a female subject for bringing about offspring with healthy neural development are disclosed. These aspects include administering to the female subject a bacterial composition comprising spore-forming bacteria of the order Clostridiales, in which the bacterial composition is administered at least once during a two-month period that ends with the day of an expected or possible conception for the female subject.

In some aspects, methods of selecting a female subject for conditioning to foster healthy neural development in offspring are disclosed. These aspects include determining that a compound in a serum sample from the female subject, bacteria of the order Clostridiales in a fecal sample from the female subject, or both satisfy an applicable criterion, and selecting the female subject for conditioning to foster healthy neural development in offspring. The compound is 2-(4-hydroxyphenyl)propionate; 3-(3-hydroxyphenyl)propionate sulfate; 3-indoxyl sulfate; 3-phenylpropionate (hydrocinnamate); 7-ketodeoxycholate; alpha-ketoglutaramate; alpha-muricholate; beta-muricholate; biotin; deoxycholate; hippurate; imidazole propionate; indolepropionate; N,N,N-trimethyl-5-aminovalerate; p-cresol sulfate; phenylpropionylglycine; pyrraline; stachydrine; taurodeoxycholate; taurohyodeoxycholic acid; trimethylamine N-oxide; ursodeoxycholate; or a combination thereof. The applicable criterion for the compound (or a combination of compounds) is for the compound to have a level in a serum sample from the female subject that is at most 10%, 20%, 30%, 40%, 50%, 60%, or 70% of its level in a control serum sample representative of a healthy female subject. The applicable criterion for the bacteria of the order Clostridiales is for them to have a total level in a fecal sample from the female subject that is at most 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 14%, 16%, 18%, or 20% of their total level in a control fecal sample representative of a healthy female subject.

In some embodiments of these aspects related to selecting a female subject, the methods further include administering to the female subject a composition that comprises trimethylamine N-oxide (TMAO), 5-aminovalerate (5-AV), imidazole propionate (IP), hippurate (HIP), or a combination thereof; a bacterial composition that comprises spore-forming bacteria of the order Clostridiales; or a combination thereof.

In certain embodiments of these aspects related to selecting a female subject, the compound is 3-indoxyl sulfate; biotin; hippurate; imidazole propionate; N,N,N-trimethyl-5-aminovalerate; pyrraline; stachydrine; trimethylamine N-oxide; or a combination thereof; and the bacteria of the order Clostridiales are bacteria of the genus Clostridium, genus Dehalobacterium, genus Ruminococcus, genus Coprococcus, genus Dorea, genus Oscillospira, or a combination thereof. In some of these methods, liquid chromatography-mass spectrometry is used to determine a level for the compound. In some embodiments, 16S rDNA sequencing is used to determine a total level for the bacteria.

In certain embodiments disclosed herein, the unborn baby or offspring is a fetus more than eight weeks after conception.

In certain embodiments, the present invention provides methods comprising administering to a maternal subject gestating a fetus a composition comprising: a compound selected from trimethylamine-N-oxide (TMAO), N,N,N-trimethyl-5-aminovaleric acid (TMAV), imidazole propionic acid (IP), 3-indoxyl sulfuric acid (3-IS), hippuric acid (HIP), perfluorooctanesulfonic acid (PFOS), 3-sulfo-L-alanine, alpha-ketoglutaramic acid, 4-hydroxyglutamic acid, extoine, stachydrine, biotin, pyroglutamine, chiro-inositol, N1-methyl-2-pyridone-5-carboxamide, O-sulfo-L-tyrosine, 2′deoxyuridine, pyrraline, N-delta-acetylornithine, phenyl sulfuric acid, phenylacetylglycine, anserine, homostachydrine, serine, N1-methyl-4-pyridone-3-carboxamide, methyl glycopyranoside (alpha+beta), 3-carboxy-1-methylpyridin-1-ium, hypotaurine, 1,5-anhydroglucitol (1,5-AG), 1-oleoyl-2-linoleoyl-GPC (18:1/18:2), methionine, glutamine, malic acid, pantothenic acid, 5,6-dihydrouridine, ceramide (d18:1/17:0 d17:1/18.0) N6-methyllysine, allantoin, N2-acetyllysine, N-acetylglutamine, stearoylcarnitine (C18), arachidoylcarnitine (C20), arabitol, xylitol, 1-methylhistamine, xanthosine, xanthine, 1-ribosyl-imidazoleacetic acid, 5-methyl-2′-deoxycytidine, sphingomyelin (d18:0/20:0, d16:0/22:0), 1-methyl-4-imidazoleacetic acid, inosine 5′-monophosphoric acid (IMP), 1,2-distearoyl-GPC (18:0/18:0), 3-methylcytidine, pipecolate, N-stearoyl-sphingadienine (d18:2/18:0), homoserine, carnosine, 1-palmitoyl-GPI (16:0), 1-stearoyl-GPI (18:0), N6-succinyladenosine, 2′-deoxycytidine, stearoyl-docosahexaenoyl-glycerol (18:0/22:6), trigonelline (N′-methylnicotinate), hydroxyasparagine, gamma-glutamylglutamic acid, 2-palmitoylglycerol (16:0), ceramide (d18:1/17:0, d17:1/18:0), thiamin (Vitamin B1), N6-methyllysine, N6,N6-dimethyllysine, 3-hydroxy-3-methylglutaric acid, campesterol, allantoin, stachydrine, N2-acetyllysine, phenyllactic acid (PLA), gamma-glutamyltryptophan, N-palmitoyl-sphingosine (d18:1/16:0), O-sulfo-L-tyrosine, indolelactic acid, gamma-glutamylglutamine, N-acetylglucosamine 6-phosphoric acid, 1-oleoyl-GPS (18:1), 3-hydroxypalmitoylcarnitine, myo-inositol, behenoyl sphingomyelin (d18:1/22:0), maltotetraose, maltotriose, N-acetylglucosamine/N-acetylgalactosamine, N1-methyladenosine, uracil, 1-oleoyl-GPI (18:1), sphingomyelin (d18:1/17:0, d17:1/18:0, d19:1/16:0), 3-ureidopropionic acid, 5-oxoproline, gamma-glutamyltyrosine, 1-(1-enyl-stearoyl)-GPE (P-18:0), cytidine 2′,3′-cyclic monophosphoric acid, 2′-deoxyguanosine 5′-monophosphoric acid (dGMP), thymidine, N6,N6,N6-trimethyllysine, 1-palmitoyl-GPC (16:0), 1-(1-enyl-palmitoyl)-GPE (P-16:0), N-stearoyl-sphinganine (d18:0/18:0), N-arachidoyl-sphingosine (d18:1/20:0), 3′-dephosphocoenzyme A, 5-hydroxylysine, arabonic acid/xylonic acid, 1-palmitoyl-2-docosahexaenoyl-GPC (16:0/22:6), glutamine, 1-(1-enyl-palmitoyl)-2-palmitoyl-GPC (P-16:0/16:0), N-behenoyl-sphingadienine (d18:2/22:0), xylulose 5-phosphoric acid, 1-oleoyl-GPC (18:1), 1-stearoyl-GPE (18:0), glycerol 3-phosphoric acid, N-stearoyl-sphingosine (d18:1/18:0), 7-methylguanine, N2,N2-dimethylguanosine, N-acetylglutamine, methionine, pro-hydroxy-pro, dihydroxyacetone phosphoric acid (DHAP), 1-stearoyl-2-docosahexaenoyl-GPC (18:0/22:6), sphingomyelin (d18:1/20:0, d16:1/22:0), uric acid, adenylosuccinic acid, cystathionine, spermine, mannitol/sorbitol, 2-hydroxyadipic acid, N-palmitoyl-sphinganine (d18:0/16:0), sphingomyelin (d18:0/18:0, d19:0/17:0), sphingomyelin (d18:1/24:1, d18:2/24:0), alpha-hydroxyisovaleric acid, citrulline, ribulonic acid/xylulonic acid, succinylcarnitine (C4-DC), ceramide (d16:1/24:1, d18:1/22:1), hypoxanthine, 5,6-dihydrouridine, gamma-aminobutyric acid (GABA), oleoyl ethanolamide, choline, 1-palmitoyl-GPE (16:0), palmitoyl-linoleoyl-glycerol (16:0/18:2), ceramide (d18:2/24:1, d18:1/24:2), cholesterol, 2′-O-methylcytidine, nicotinamide riboside, pantothenic acid, pyridoxal, N-acetylaspartic acid (NAA), C-glycosyltryptophan, methionine sulfoxide, spermidine, 1-palmitoyl-2-oleoyl-GPG (16:0/18:1), lignoceroyl sphingomyelin (d18:1/24:0), desmosterol, N1-methylinosine, cytidine, N-acetyl-aspartyl-glutamic acid (NAAG), sedoheptulose, galactonic acid, cytidine 5′-monophospho-N-acetylneuraminic acid, glycerophosphoinositol, uridine, salicylic acid, N-acetylglutamic acid, gamma-glutamyl-epsilon-lysine, glycerophosphoserine, 1-stearoyl-2-oleoyl-GPE (18:0/18:1), beta-alanine, 5-methylcytidine, methylphosphoric acid, imidazole lactic acid, sedoheptulose-7-phosphoric acid, 1-palmitoyl-2-stearoyl-GPE (16:0/18:0), guanosine 5′-diphosphoric acid (GDP), 3-ureidoisobutyric acid, tryptophan, isoleucine, methyl succinic acid, S-adenosylmethionine (SAM), taurine, gamma-glutamylthreonine, arabitol/xylitol, erythronic acid, fumaric acid, stearoylcarnitine (C18), deoxycarnitine, cytidine 5′-diphosphocholine, 1-stearoyl-2-arachidonoyl-GPC (18:0/20:4), 1-stearoyl-2-arachidonoyl-GPI (18:0/20:4), glycerophosphoglycerol, N6-carbamoylthreonyladenosine, flavin adenine dinucleotide (FAD), 2-oxoarginine, lactic acid, gulonic acid, phenylalanine, 3-(4-hydroxyphenyl)lactic acid, 2-hydroxyglutaric acid, palmitoleoyl ethanolamide, 1-palmitoyl-2-stearoyl-GPC (16:0/18:0), 1-stearoyl-2-oleoyl-GPC (18:0/18:1), palmitoyl-oleoyl-glycerol (16:0/18:1), betaine, N-acetylneuraminic acid, malic acid, phosphoethanolamine, 1-myristoyl-2-arachidonoyl-GPC (14:0/20:4), beta-citrylglutamic acid, 1-methylhistidine, leucine, ethylmalonic acid, prolylglycine, stearoyl-arachidonoyl-glycerol (18:0/20:4), orotidine, 5-(galactosylhydroxy)-L-lysine, N-acetylglucosaminylasparagine, eicosenoylcarnitine (C20:1), cytidine-5′-diphosphoethanolamine, glycosyl-N-stearoyl-sphingosine (d18:1/18:0), palmitoyl dihydrosphingomyelin (d18:0/16:0), sphingosine, inosine, guanosine 5′-monophosphoric acid (5′-GMP), dimethylglycine, N-acetylalanine, aspartic acid, creatine, ribitol, 2-methylcitric acid/homocitric acid, arachidoylcarnitine (C20), S-methylglutathione, 1-palmitoyl-2-arachidonoyl-GPC (16:0/20:4n6), stearoyl sphingomyelin (d18:1/18:0), nicotinamide, N-formylmethionine, UDP-N-acetylglucosamine/galactosamine, glucoronic acid, 1,2-dipalmitoyl-GPE (16:0/16:0), pseudouridine, alanine, glutamic acid, 1-myristoyl-2-palmitoyl-GPC (14:0/16:0), 1,2-dipalmitoyl-GPC (16:0/16:0), 1-palmitoyl-2-oleoyl-GPC (16:0/18:1), sphingomyelin (d18:1/22:1, d18:2/22:0, d16:1/24:1), glycerophosphoethanolamine, 1-palmitoyl-2-palmitoleoyl-GPC (16:0/16:1), UDP-glucuronic acid, and 1-methylnicotinamide, or a salt thereof, or a combination thereof; and/or one or more bacterial species found in a maternal microbiome.

In certain embodiments, the present invention provides methods comprising administering to a female subject a composition comprising: a compound selected from trimethylamine-N-oxide (TMAO), N,N,N-trimethyl-5-aminovaleric acid (TMAV), imidazole propionic acid (IP), 3-indoxyl sulfuric acid (3-IS), hippuric acid (HIP), perfluorooctanesulfonic acid (PFOS), 3-sulfo-L-alanine, alpha-ketoglutaramic acid, 4-hydroxyglutamic acid, extoine, stachydrine, biotin, pyroglutamine, chiro-inositol, N1-methyl-2-pyridone-5-carboxamide, O-sulfo-L-tyrosine, 2′deoxyuridine, pyrraline, N-delta-acetylornithine, phenyl sulfuric acid, phenylacetylglycine, anserine, homostachydrine, serine, N1-methyl-4-pyridone-3-carboxamide, methyl glycopyranoside (alpha+beta), 3-carboxy-1-methylpyridin-1-ium, hypotaurine, 1,5-anhydroglucitol (1,5-AG), 1-oleoyl-2-linoleoyl-GPC (18:1/18:2), methionine, glutamine, malic acid, pantothenic acid, 5,6-dihydrouridine, ceramide (d18:1/17:0 d17:1/18.0) N6-methyllysine, allantoin, N2-acetyllysine, N-acetylglutamine, stearoylcarnitine (C18), arachidoylcarnitine (C20), arabitol, xylitol, 1-methylhistamine, xanthosine, xanthine, 1-ribosyl-imidazoleacetic acid, 5-methyl-2′-deoxycytidine, sphingomyelin (d18:0/20:0, d16:0/22:0), 1-methyl-4-imidazoleacetic acid, inosine 5′-monophosphoric acid (IMP), 1,2-distearoyl-GPC (18:0/18:0), 3-methylcytidine, pipecolate, N-stearoyl-sphingadienine (d18:2/18:0), homoserine, carnosine, 1-palmitoyl-GPI (16:0), 1-stearoyl-GPI (18:0), N6-succinyladenosine, 2′-deoxycytidine, stearoyl-docosahexaenoyl-glycerol (18:0/22:6), trigonelline (N′-methylnicotinate), hydroxyasparagine, gamma-glutamylglutamic acid, 2-palmitoylglycerol (16:0), ceramide (d18:1/17:0, d17:1/18:0), thiamin (Vitamin B1), N6-methyllysine, N6,N6-dimethyllysine, 3-hydroxy-3-methylglutaric acid, campesterol, allantoin, stachydrine, N2-acetyllysine, phenyllactic acid (PLA), gamma-glutamyltryptophan, N-palmitoyl-sphingosine (d18:1/16:0), O-sulfo-L-tyrosine, indolelactic acid, gamma-glutamylglutamine, N-acetylglucosamine 6-phosphoric acid, 1-oleoyl-GPS (18:1), 3-hydroxypalmitoylcarnitine, myo-inositol, behenoyl sphingomyelin (d18:1/22:0), maltotetraose, maltotriose, N-acetylglucosamine/N-acetylgalactosamine, N1-methyladenosine, uracil, 1-oleoyl-GPI (18:1), sphingomyelin (d18:1/17:0, d17:1/18:0, d19:1/16:0), 3-ureidopropionic acid, 5-oxoproline, gamma-glutamyltyrosine, 1-(1-enyl-stearoyl)-GPE (P-18:0), cytidine 2′,3′-cyclic monophosphoric acid, 2′-deoxyguanosine 5′-monophosphoric acid (dGMP), thymidine, N6,N6,N6-trimethyllysine, 1-palmitoyl-GPC (16:0), 1-(1-enyl-palmitoyl)-GPE (P-16:0), N-stearoyl-sphinganine (d18:0/18:0), N-arachidoyl-sphingosine (d18:1/20:0), 3′-dephosphocoenzyme A, 5-hydroxylysine, arabonic acid/xylonic acid, 1-palmitoyl-2-docosahexaenoyl-GPC (16:0/22:6), glutamine, 1-(1-enyl-palmitoyl)-2-palmitoyl-GPC (P-16:0/16:0), N-behenoyl-sphingadienine (d18:2/22:0), xylulose 5-phosphoric acid, 1-oleoyl-GPC (18:1), 1-stearoyl-GPE (18:0), glycerol 3-phosphoric acid, N-stearoyl-sphingosine (d18:1/18:0), 7-methylguanine, N2,N2-dimethylguanosine, N-acetylglutamine, methionine, pro-hydroxy-pro, dihydroxyacetone phosphoric acid (DHAP), 1-stearoyl-2-docosahexaenoyl-GPC (18:0/22:6), sphingomyelin (d18:1/20:0, d16:1/22:0), uric acid, adenylosuccinic acid, cystathionine, spermine, mannitol/sorbitol, 2-hydroxyadipic acid, N-palmitoyl-sphinganine (d18:0/16:0), sphingomyelin (d18:0/18:0, d19:0/17:0), sphingomyelin (d18:1/24:1, d18:2/24:0), alpha-hydroxyisovaleric acid, citrulline, ribulonic acid/xylulonic acid, succinylcarnitine (C4-DC), ceramide (d16:1/24:1, d18:1/22:1), hypoxanthine, 5,6-dihydrouridine, gamma-aminobutyric acid (GABA), oleoyl ethanolamide, choline, 1-palmitoyl-GPE (16:0), palmitoyl-linoleoyl-glycerol (16:0/18:2), ceramide (d18:2/24:1, d18:1/24:2), cholesterol, 2′-O-methylcytidine, nicotinamide riboside, pantothenic acid, pyridoxal, N-acetylaspartic acid (NAA), C-glycosyltryptophan, methionine sulfoxide, spermidine, 1-palmitoyl-2-oleoyl-GPG (16:0/18:1), lignoceroyl sphingomyelin (d18:1/24:0), desmosterol, N1-methylinosine, cytidine, N-acetyl-aspartyl-glutamic acid (NAAG), sedoheptulose, galactonic acid, cytidine 5′-monophospho-N-acetylneuraminic acid, glycerophosphoinositol, uridine, salicylic acid, N-acetylglutamic acid, gamma-glutamyl-epsilon-lysine, glycerophosphoserine, 1-stearoyl-2-oleoyl-GPE (18:0/18:1), beta-alanine, 5-methylcytidine, methylphosphoric acid, imidazole lactic acid, sedoheptulose-7-phosphoric acid, 1-palmitoyl-2-stearoyl-GPE (16:0/18:0), guanosine 5′-diphosphoric acid (GDP), 3-ureidoisobutyric acid, tryptophan, isoleucine, methyl succinic acid, S-adenosylmethionine (SAM), taurine, gamma-glutamylthreonine, arabitol/xylitol, erythronic acid, fumaric acid, stearoylcarnitine (C18), deoxycarnitine, cytidine 5′-diphosphocholine, 1-stearoyl-2-arachidonoyl-GPC (18:0/20:4), 1-stearoyl-2-arachidonoyl-GPI (18:0/20:4), glycerophosphoglycerol, N6-carbamoylthreonyladenosine, flavin adenine dinucleotide (FAD), 2-oxoarginine, lactic acid, gulonic acid, phenylalanine, 3-(4-hydroxyphenyl)lactic acid, 2-hydroxyglutaric acid, palmitoleoyl ethanolamide, 1-palmitoyl-2-stearoyl-GPC (16:0/18:0), 1-stearoyl-2-oleoyl-GPC (18:0/18:1), palmitoyl-oleoyl-glycerol (16:0/18:1), betaine, N-acetylneuraminic acid, malic acid, phosphoethanolamine, 1-myristoyl-2-arachidonoyl-GPC (14:0/20:4), beta-citrylglutamic acid, 1-methylhistidine, leucine, ethylmalonic acid, prolylglycine, stearoyl-arachidonoyl-glycerol (18:0/20:4), orotidine, 5-(galactosylhydroxy)-L-lysine, N-acetylglucosaminylasparagine, eicosenoylcarnitine (C20:1), cytidine-5′-diphosphoethanolamine, glycosyl-N-stearoyl-sphingosine (d18:1/18:0), palmitoyl dihydrosphingomyelin (d18:0/16:0), sphingosine, inosine, guanosine 5′-monophosphoric acid (5′-GMP), dimethylglycine, N-acetylalanine, aspartic acid, creatine, ribitol, 2-methylcitric acid/homocitric acid, arachidoylcarnitine (C20), S-methylglutathione, 1-palmitoyl-2-arachidonoyl-GPC (16:0/20:4n6), stearoyl sphingomyelin (d18:1/18:0), nicotinamide, N-formylmethionine, UDP-N-acetylglucosamine/galactosamine, glucoronic acid, 1,2-dipalmitoyl-GPE (16:0/16:0), pseudouridine, alanine, glutamic acid, 1-myristoyl-2-palmitoyl-GPC (14:0/16:0), 1,2-dipalmitoyl-GPC (16:0/16:0), 1-palmitoyl-2-oleoyl-GPC (16:0/18:1), sphingomyelin (d18:1/22:1, d18:2/22:0, d16:1/24:1), glycerophosphoethanolamine, 1-palmitoyl-2-palmitoleoyl-GPC (16:0/16:1), UDP-glucuronic acid, and 1-methylnicotinamide, or a salt thereof, or a combination thereof; and/or one or more bacterial species found in a maternal microbiome; wherein the female subject is a fertile, ovulating female subject or a female subject seeking to implant an embryo.

Also disclosed herein are methods of inhibiting development of a disease or disorder in a fetus.

In certain embodiments, the present invention provides methods comprising administering to a maternal subject gestating the fetus, a composition comprising: a compound selected from trimethylamine-N-oxide (TMAO), N,N,N-trimethyl-5-aminovaleric acid (TMAV), imidazole propionic acid (IP), 3-indoxyl sulfuric acid (3-IS), hippuric acid (HIP), perfluorooctanesulfonic acid (PFOS), 3-sulfo-L-alanine, alpha-ketoglutaramic acid, 4-hydroxyglutamic acid, extoine, stachydrine, biotin, pyroglutamine, chiro-inositol, N1-methyl-2-pyridone-5-carboxamide, O-sulfo-L-tyrosine, 2′deoxyuridine, pyrraline, N-delta-acetylornithine, phenyl sulfuric acid, phenylacetylglycine, anserine, homostachydrine, serine, N1-methyl-4-pyridone-3-carboxamide, methyl glycopyranoside (alpha+beta), 3-carboxy-1-methylpyridin-1-ium, hypotaurine, 1,5-anhydroglucitol (1,5-AG), 1-oleoyl-2-linoleoyl-GPC (18:1/18:2), methionine, glutamine, malic acid, pantothenic acid, 5,6-dihydrouridine, ceramide (d18:1/17:0 d17:1/18.0) N6-methyllysine, allantoin, N2-acetyllysine, N-acetylglutamine, stearoylcarnitine (C18), arachidoylcarnitine (C20), arabitol, xylitol, 1-methylhistamine, xanthosine, xanthine, 1-ribosyl-imidazoleacetic acid, 5-methyl-2′-deoxycytidine, sphingomyelin (d18:0/20:0, d16:0/22:0), 1-methyl-4-imidazoleacetic acid, inosine 5′-monophosphoric acid (IMP), 1,2-distearoyl-GPC (18:0/18:0), 3-methylcytidine, pipecolate, N-stearoyl-sphingadienine (d18:2/18:0), homoserine, carnosine, 1-palmitoyl-GPI (16:0), 1-stearoyl-GPI (18:0), N6-succinyladenosine, 2′-deoxycytidine, stearoyl-docosahexaenoyl-glycerol (18:0/22:6), trigonelline (N′-methylnicotinate), hydroxyasparagine, gamma-glutamylglutamic acid, 2-palmitoylglycerol (16:0), ceramide (d18:1/17:0, d17:1/18:0), thiamin (Vitamin B1), N6-methyllysine, N6,N6-dimethyllysine, 3-hydroxy-3-methylglutaric acid, campesterol, allantoin, stachydrine, N2-acetyllysine, phenyllactic acid (PLA), gamma-glutamyltryptophan, N-palmitoyl-sphingosine (d18:1/16:0), O-sulfo-L-tyrosine, indolelactic acid, gamma-glutamylglutamine, N-acetylglucosamine 6-phosphoric acid, 1-oleoyl-GPS (18:1), 3-hydroxypalmitoylcarnitine, myo-inositol, behenoyl sphingomyelin (d18:1/22:0), maltotetraose, maltotriose, N-acetylglucosamine/N-acetylgalactosamine, N1-methyladenosine, uracil, 1-oleoyl-GPI (18:1), sphingomyelin (d18:1/17:0, d17:1/18:0, d19:1/16:0), 3-ureidopropionic acid, 5-oxoproline, gamma-glutamyltyrosine, 1-(1-enyl-stearoyl)-GPE (P-18:0), cytidine 2′,3′-cyclic monophosphoric acid, 2′-deoxyguanosine 5′-monophosphoric acid (dGMP), thymidine, N6,N6,N6-trimethyllysine, 1-palmitoyl-GPC (16:0), 1-(1-enyl-palmitoyl)-GPE (P-16:0), N-stearoyl-sphinganine (d18:0/18:0), N-arachidoyl-sphingosine (d18:1/20:0), 3′-dephosphocoenzyme A, 5-hydroxylysine, arabonic acid/xylonic acid, 1-palmitoyl-2-docosahexaenoyl-GPC (16:0/22:6), glutamine, 1-(1-enyl-palmitoyl)-2-palmitoyl-GPC (P-16:0/16:0), N-behenoyl-sphingadienine (d18:2/22:0), xylulose 5-phosphoric acid, 1-oleoyl-GPC (18:1), 1-stearoyl-GPE (18:0), glycerol 3-phosphoric acid, N-stearoyl-sphingosine (d18:1/18:0), 7-methylguanine, N2,N2-dimethylguanosine, N-acetylglutamine, methionine, pro-hydroxy-pro, dihydroxyacetone phosphoric acid (DHAP), 1-stearoyl-2-docosahexaenoyl-GPC (18:0/22:6), sphingomyelin (d18:1/20:0, d16:1/22:0), uric acid, adenylosuccinic acid, cystathionine, spermine, mannitol/sorbitol, 2-hydroxyadipic acid, N-palmitoyl-sphinganine (d18:0/16:0), sphingomyelin (d18:0/18:0, d19:0/17:0), sphingomyelin (d18:1/24:1, d18:2/24:0), alpha-hydroxyisovaleric acid, citrulline, ribulonic acid/xylulonic acid, succinylcarnitine (C4-DC), ceramide (d16:1/24:1, d18:1/22:1), hypoxanthine, 5,6-dihydrouridine, gamma-aminobutyric acid (GABA), oleoyl ethanolamide, choline, 1-palmitoyl-GPE (16:0), palmitoyl-linoleoyl-glycerol (16:0/18:2), ceramide (d18:2/24:1, d18:1/24:2), cholesterol, 2′-O-methylcytidine, nicotinamide riboside, pantothenic acid, pyridoxal, N-acetylaspartic acid (NAA), C-glycosyltryptophan, methionine sulfoxide, spermidine, 1-palmitoyl-2-oleoyl-GPG (16:0/18:1), lignoceroyl sphingomyelin (d18:1/24:0), desmosterol, N1-methylinosine, cytidine, N-acetyl-aspartyl-glutamic acid (NAAG), sedoheptulose, galactonic acid, cytidine 5′-monophospho-N-acetylneuraminic acid, glycerophosphoinositol, uridine, salicylic acid, N-acetylglutamic acid, gamma-glutamyl-epsilon-lysine, glycerophosphoserine, 1-stearoyl-2-oleoyl-GPE (18:0/18:1), beta-alanine, 5-methylcytidine, methylphosphoric acid, imidazole lactic acid, sedoheptulose-7-phosphoric acid, 1-palmitoyl-2-stearoyl-GPE (16:0/18:0), guanosine 5′-diphosphoric acid (GDP), 3-ureidoisobutyric acid, tryptophan, isoleucine, methyl succinic acid, S-adenosylmethionine (SAM), taurine, gamma-glutamylthreonine, arabitol/xylitol, erythronic acid, fumaric acid, stearoylcarnitine (C18), deoxycarnitine, cytidine 5′-diphosphocholine, 1-stearoyl-2-arachidonoyl-GPC (18:0/20:4), 1-stearoyl-2-arachidonoyl-GPI (18:0/20:4), glycerophosphoglycerol, N6-carbamoylthreonyladenosine, flavin adenine dinucleotide (FAD), 2-oxoarginine, lactic acid, gulonic acid, phenylalanine, 3-(4-hydroxyphenyl)lactic acid, 2-hydroxyglutaric acid, palmitoleoyl ethanolamide, 1-palmitoyl-2-stearoyl-GPC (16:0/18:0), 1-stearoyl-2-oleoyl-GPC (18:0/18:1), palmitoyl-oleoyl-glycerol (16:0/18:1), betaine, N-acetylneuraminic acid, malic acid, phosphoethanolamine, 1-myristoyl-2-arachidonoyl-GPC (14:0/20:4), beta-citrylglutamic acid, 1-methylhistidine, leucine, ethylmalonic acid, prolylglycine, stearoyl-arachidonoyl-glycerol (18:0/20:4), orotidine, 5-(galactosylhydroxy)-L-lysine, N-acetylglucosaminylasparagine, eicosenoylcarnitine (C20:1), cytidine-5′-diphosphoethanolamine, glycosyl-N-stearoyl-sphingosine (d18:1/18:0), palmitoyl dihydrosphingomyelin (d18:0/16:0), sphingosine, inosine, guanosine 5′-monophosphoric acid (5′-GMP), dimethylglycine, N-acetylalanine, aspartic acid, creatine, ribitol, 2-methylcitric acid/homocitric acid, arachidoylcarnitine (C20), S-methylglutathione, 1-palmitoyl-2-arachidonoyl-GPC (16:0/20:4n6), stearoyl sphingomyelin (d18:1/18:0), nicotinamide, N-formylmethionine, UDP-N-acetylglucosamine/galactosamine, glucoronic acid, 1,2-dipalmitoyl-GPE (16:0/16:0), pseudouridine, alanine, glutamic acid, 1-myristoyl-2-palmitoyl-GPC (14:0/16:0), 1,2-dipalmitoyl-GPC (16:0/16:0), 1-palmitoyl-2-oleoyl-GPC (16:0/18:1), sphingomyelin (d18:1/22:1, d18:2/22:0, d16:1/24:1), glycerophosphoethanolamine, 1-palmitoyl-2-palmitoleoyl-GPC (16:0/16:1), UDP-glucuronic acid, and 1-methylnicotinamide, or a salt thereof, or a combination thereof; and/or one or more bacterial species found in a maternal microbiome.

In certain embodiments, the present invention provides methods comprising administering to a female subject a composition comprising: a compound selected from trimethylamine-N-oxide (TMAO), N,N,N-trimethyl-5-aminovaleric acid (TMAV), imidazole propionic acid (IP), 3-indoxyl sulfuric acid (3-IS), hippuric acid (HIP), perfluorooctanesulfonic acid (PFOS), 3-sulfo-L-alanine, alpha-ketoglutaramic acid, 4-hydroxyglutamic acid, extoine, stachydrine, biotin, pyroglutamine, chiro-inositol, N1-methyl-2-pyridone-5-carboxamide, O-sulfo-L-tyrosine, 2′deoxyuridine, pyrraline, N-delta-acetylornithine, phenyl sulfuric acid, phenylacetylglycine, anserine, homostachydrine, serine, N1-methyl-4-pyridone-3-carboxamide, methyl glycopyranoside (alpha+beta), 3-carboxy-1-methylpyridin-1-ium, hypotaurine, 1,5-anhydroglucitol (1,5-AG), 1-oleoyl-2-linoleoyl-GPC (18:1/18:2), methionine, glutamine, malic acid, pantothenic acid, 5,6-dihydrouridine, ceramide (d18:1/17:0 d17:1/18.0) N6-methyllysine, allantoin, N2-acetyllysine, N-acetylglutamine, stearoylcarnitine (C18), arachidoylcarnitine (C20), arabitol, xylitol, 1-methylhistamine, xanthosine, xanthine, 1-ribosyl-imidazoleacetic acid, 5-methyl-2′-deoxycytidine, sphingomyelin (d18:0/20:0, d16:0/22:0), 1-methyl-4-imidazoleacetic acid, inosine 5′-monophosphoric acid (IMP), 1,2-distearoyl-GPC (18:0/18:0), 3-methylcytidine, pipecolate, N-stearoyl-sphingadienine (d18:2/18:0), homoserine, carnosine, 1-palmitoyl-GPI (16:0), 1-stearoyl-GPI (18:0), N6-succinyladenosine, 2′-deoxycytidine, stearoyl-docosahexaenoyl-glycerol (18:0/22:6), trigonelline (N′-methylnicotinate), hydroxyasparagine, gamma-glutamylglutamic acid, 2-palmitoylglycerol (16:0), ceramide (d18:1/17:0, d17:1/18:0), thiamin (Vitamin B1), N6-methyllysine, N6,N6-dimethyllysine, 3-hydroxy-3-methylglutaric acid, campesterol, allantoin, stachydrine, N2-acetyllysine, phenyllactic acid (PLA), gamma-glutamyltryptophan, N-palmitoyl-sphingosine (d18:1/16:0), O-sulfo-L-tyrosine, indolelactic acid, gamma-glutamylglutamine, N-acetylglucosamine 6-phosphoric acid, 1-oleoyl-GPS (18:1), 3-hydroxypalmitoylcarnitine, myo-inositol, behenoyl sphingomyelin (d18:1/22:0), maltotetraose, maltotriose, N-acetylglucosamine/N-acetylgalactosamine, N1-methyladenosine, uracil, 1-oleoyl-GPI (18:1), sphingomyelin (d18:1/17:0, d17:1/18:0, d19:1/16:0), 3-ureidopropionic acid, 5-oxoproline, gamma-glutamyltyrosine, 1-(1-enyl-stearoyl)-GPE (P-18:0), cytidine 2′,3′-cyclic monophosphoric acid, 2′-deoxyguanosine 5′-monophosphoric acid (dGMP), thymidine, N6,N6,N6-trimethyllysine, 1-palmitoyl-GPC (16:0), 1-(1-enyl-palmitoyl)-GPE (P-16:0), N-stearoyl-sphinganine (d18:0/18:0), N-arachidoyl-sphingosine (d18:1/20:0), 3′-dephosphocoenzyme A, 5-hydroxylysine, arabonic acid/xylonic acid, 1-palmitoyl-2-docosahexaenoyl-GPC (16:0/22:6), glutamine, 1-(1-enyl-palmitoyl)-2-palmitoyl-GPC (P-16:0/16:0), N-behenoyl-sphingadienine (d18:2/22:0), xylulose 5-phosphoric acid, 1-oleoyl-GPC (18:1), 1-stearoyl-GPE (18:0), glycerol 3-phosphoric acid, N-stearoyl-sphingosine (d18:1/18:0), 7-methylguanine, N2,N2-dimethylguanosine, N-acetylglutamine, methionine, pro-hydroxy-pro, dihydroxyacetone phosphoric acid (DHAP), 1-stearoyl-2-docosahexaenoyl-GPC (18:0/22:6), sphingomyelin (d18:1/20:0, d16:1/22:0), uric acid, adenylosuccinic acid, cystathionine, spermine, mannitol/sorbitol, 2-hydroxyadipic acid, N-palmitoyl-sphinganine (d18:0/16:0), sphingomyelin (d18:0/18:0, d19:0/17:0), sphingomyelin (d18:1/24:1, d18:2/24:0), alpha-hydroxyisovaleric acid, citrulline, ribulonic acid/xylulonic acid, succinylcarnitine (C4-DC), ceramide (d16:1/24:1, d18:1/22:1), hypoxanthine, 5,6-dihydrouridine, gamma-aminobutyric acid (GABA), oleoyl ethanolamide, choline, 1-palmitoyl-GPE (16:0), palmitoyl-linoleoyl-glycerol (16:0/18:2), ceramide (d18:2/24:1, d18:1/24:2), cholesterol, 2′-O-methylcytidine, nicotinamide riboside, pantothenic acid, pyridoxal, N-acetylaspartic acid (NAA), C-glycosyltryptophan, methionine sulfoxide, spermidine, 1-palmitoyl-2-oleoyl-GPG (16:0/18:1), lignoceroyl sphingomyelin (d18:1/24:0), desmosterol, N1-methylinosine, cytidine, N-acetyl-aspartyl-glutamic acid (NAAG), sedoheptulose, galactonic acid, cytidine 5′-monophospho-N-acetylneuraminic acid, glycerophosphoinositol, uridine, salicylic acid, N-acetylglutamic acid, gamma-glutamyl-epsilon-lysine, glycerophosphoserine, 1-stearoyl-2-oleoyl-GPE (18:0/18:1), beta-alanine, 5-methylcytidine, methylphosphoric acid, imidazole lactic acid, sedoheptulose-7-phosphoric acid, 1-palmitoyl-2-stearoyl-GPE (16:0/18:0), guanosine 5′-diphosphoric acid (GDP), 3-ureidoisobutyric acid, tryptophan, isoleucine, methyl succinic acid, S-adenosylmethionine (SAM), taurine, gamma-glutamylthreonine, arabitol/xylitol, erythronic acid, fumaric acid, stearoylcarnitine (C18), deoxycarnitine, cytidine 5′-diphosphocholine, 1-stearoyl-2-arachidonoyl-GPC (18:0/20:4), 1-stearoyl-2-arachidonoyl-GPI (18:0/20:4), glycerophosphoglycerol, N6-carbamoylthreonyladenosine, flavin adenine dinucleotide (FAD), 2-oxoarginine, lactic acid, gulonic acid, phenylalanine, 3-(4-hydroxyphenyl)lactic acid, 2-hydroxyglutaric acid, palmitoleoyl ethanolamide, 1-palmitoyl-2-stearoyl-GPC (16:0/18:0), 1-stearoyl-2-oleoyl-GPC (18:0/18:1), palmitoyl-oleoyl-glycerol (16:0/18:1), betaine, N-acetylneuraminic acid, malic acid, phosphoethanolamine, 1-myristoyl-2-arachidonoyl-GPC (14:0/20:4), beta-citrylglutamic acid, 1-methylhistidine, leucine, ethylmalonic acid, prolylglycine, stearoyl-arachidonoyl-glycerol (18:0/20:4), orotidine, 5-(galactosylhydroxy)-L-lysine, N-acetylglucosaminylasparagine, eicosenoylcarnitine (C20:1), cytidine-5′-diphosphoethanolamine, glycosyl-N-stearoyl-sphingosine (d18:1/18:0), palmitoyl dihydrosphingomyelin (d18:0/16:0), sphingosine, inosine, guanosine 5′-monophosphoric acid (5′-GMP), dimethylglycine, N-acetylalanine, aspartic acid, creatine, ribitol, 2-methylcitric acid/homocitric acid, arachidoylcarnitine (C20), S-methylglutathione, 1-palmitoyl-2-arachidonoyl-GPC (16:0/20:4n6), stearoyl sphingomyelin (d18:1/18:0), nicotinamide, N-formylmethionine, UDP-N-acetylglucosamine/galactosamine, glucoronic acid, 1,2-dipalmitoyl-GPE (16:0/16:0), pseudouridine, alanine, glutamic acid, 1-myristoyl-2-palmitoyl-GPC (14:0/16:0), 1,2-dipalmitoyl-GPC (16:0/16:0), 1-palmitoyl-2-oleoyl-GPC (16:0/18:1), sphingomyelin (d18:1/22:1, d18:2/22:0, d16:1/24:1), glycerophosphoethanolamine, 1-palmitoyl-2-palmitoleoyl-GPC (16:0/16:1), UDP-glucuronic acid, and 1-methylnicotinamide, or a salt thereof, or a combination thereof; and/or one or more bacterial species found in a maternal microbiome; wherein the female subject is a fertile, ovulating female subject or a female subject seeking to implant an embryo.

Such pharmaceutical preparations may be for use in treating or preventing a condition or disease as described herein.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1M: Depletion of the maternal microbiota during early gestation alters fetal brain gene expression and impairs fetal thalamocortical axonogenesis. 1A, Heatmap of differentially expressed genes in embryonic brains of E14.5 offspring from conventionally colonized (specific pathogen-free, SPF) and antibiotic-treated (ABX) dams (Wald Test, p<0.05, n=offspring from 3 dams). 1B, Representative immunofluorescence images of Netrin-G1a (magenta in the original image) and L1 (cyan in the original image) staining in embryonic brain sections (rostra′ to caudal) from E14.5 offspring of SPF, ABX and germ-free (GF) dams. Scale bar=500 μm. 1C, Average Netrin-G1a fluorescence intensity per matched area of region of interest (ROI) (“yellow-in-the-original-image” dotted lines) across 800 μm of rostral to caudal embryonic brain sections of E14.5 offspring from SPF, ABX, and GF dams. (Two-way ANOVA with Tukey's, n=offspring from 5 SPF, 7 ABX, 4 GF dams). 1D, Average L1 fluorescence normalized to control SPF ROI (“yellow-in-the-original-image” dotted lines) area across 800 μm of rostral to caudal embryonic brain sections of E14.5 offspring from SPF, ABX, and GF dams (Two-way ANOVA with Tukey's, n=offspring from 5 SPF, 7 ABX, 4 GF dams). 1E, Representative 3-D rendering of Netrin-G1a staining (purple in the original image) in cleared whole embryonic brains of E14.5 offspring from SPF, ABX, and GF dams. Th=thalamus. Scale bar=100 μm. 1F, Volume of Netrin-G1a axons from cleared whole embryonic brains of E14.5 offspring of SPF, ABX, and GF dams. (Two-way ANOVA with Tukey's (SPF vs ABX); Mann-Whitney test (SPF vs GF), n=offspring from 5 dams per group). 1G, Length of Netrin-G1a axons in cleared whole embryonic brains from E14.5 offspring of SPF, ABX, and GF dams. (Two-way ANOVA with Tukey's, n=offspring from 5 dams per group). 1H, Distance from rostral tip of Netrin-G1a staining to the cortex in cleared whole embryonic brains from E14.5 offspring of SPF, ABX, and GF dams. (Two-way ANOVA with Tukey's (SPF vs ABX); Mann-Whitney test (SPF vs GF), n=offspring from 5 dams per group). 1I, Average circumference of the Netrin-G1 a axonal bundle at the internal capsule (IC) in whole embryonic brains from E14.5 offspring of SPF, ABX, and GF dams. (Two-way ANOVA with Tukey's (SPF vs ABX); Mann-Whitney test (SPF vs GF), n=offspring from 5 dams per group). 1J, Schematic of E14.5 thalamic (Th), striatal (St) and hypothalamic (Hy) explant co-culture for axon outgrowth assay. The bar (gray colored in the original image; to the left of Th) indicates site of Th axon quantification, proximal to St. 1K, Representative TUJ1 fluorescence images of axon outgrowth from i) SPF Th explant proximal to SPF St explant (top left), ii) ABX Th explant proximal to ABX St (top right), iii) SPF Th explant proximal to ABX St explant (bottom left), iv) ABX Th explant proximal to SPF St explant (bottom right). Scale bar=250 μm. 1L, Number of axons per 200 μm of Th perimeter proximal to St explant, normalized to measurements from Th monoculture. (Two-way ANOVA with Tukey's, n=offspring from 14 SPF+SPF, 20 ABX+ABX, 10 SPF+ABX, 10 ABX+SPF dams). 1M, Length of Th axons proximal to St explant, normalized to measurements from Th monoculture. (Two-way ANOVA with Tukey's, n=offspring from 14 SPF+SPF, 20 ABX+ABX, 10 SPF+ABX, 10 ABX+SPF dams). Data are presented as mean±SEM, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, n.s.=not statistically significant.

FIGS. 2A-2D: Network analysis and qPCR validation of fetal brain RNAseq data. 2A, DAVID gene ontology analysis of differentially expressed genes from whole embryonic brains from E14.5 offspring of SPF compared to those from ABX dams (Fisher exact, q<0.05, n=offspring from 3 dams per group). 2B, Quantitative RT-PCR for NTNG1 and LRRC4C expression in whole embryonic brains from E14.5 offspring of SPF, ABX or GF dams (Two-way ANOVA with Tukey's, n=offspring from 9 SPF, 15 ABX, 10 Sp dams). 2C, STRING protein interaction network of genes downregulated in whole embryonic brains from E14.5 offspring of ABX dams compared to those from SPF dams (q<0.05, n=offspring from 3 dams). 2D, STRING protein interaction networks of genes upregulated in whole embryonic brains from E14.5 offspring of ABX dams compared to those from SPF dams (q<0.05, n=offspring from 3 dams). Data are presented as mean±SEM. *p<0.05, n.s.=not statistically significant.

FIGS. 3A-3I: Netrin-G1a thalamocortical axons in embryonic brains of E14.5 offspring from gnotobiotic dams. 3A, Reference diagrams of E14.5 coronal embryonic brain sections. 3B, Immunofluorescence images of Netrin-G1a in four independent embryonic brain sections from E14.5 offspring of SPF dams. 200 μm intervals. Scale bar: 500 μm. 3C, Immunofluorescence image of Netrin-G1a in four independent embryonic brain sections from E14.5 offspring of ABX dams. 200 μm intervals. Scale bar: 500 μm. 3D, Immunofluorescence image of Netrin-G1a in four independent embryonic brain sections from E14.5 offspring of GF dams. 200 μm intervals. Scale bar: 500 μm. 3E, Immunofluorescence image of Netrin-G1a in four independent embryonic brain sections from E14.5 offspring of dams colonized with Sp bacteria. 200 μm intervals. Scale bar: 500 μm. 3F, Average Netrin-G1 a fluorescence intensity per matched area of region of interest (ROI) (“yellow-in-the-original-image” dotted lines) in 800 μm of rostral to caudal embryonic brain sections of E14.5 offspring from SPF, ABX, GF and Sp-colonized dams. (Two-way ANOVA with Tukey's, n=offspring from 5 SPF, 7 ABX, 4 GF, 5 Sp dams). 3G, Total Netrin-G1a fluorescence intensity across 800 μm of rostral to caudal sections of E14.5 embryonic brains from SPF, ABX, GF, and Sp-colonized dams. (Two-way ANOVA with Tukey's, n=offspring from 5 SPF, 7 ABX, 4 GF, 5 Sp dams). 3H, Average area of Netrin-G1a-positive staining across 800 μm of rostral to caudal embryonic brain sections of E14.5 offspring from SPF, ABX, GF and Sp-colonized dams. (Two-way ANOVA with Tukey's, n=offspring from 5 SPF, 7 ABX, 4 GF, 5 Sp dams). 3I, Average DAPI fluorescence intensity normalized to per matched ROI (“yellow-in-the-original-image” dotted lines) in 800 μm of rostral to caudal embryonic brain sections of E14.5 offspring from SPF, ABX, GF and Sp-colonized dams. (Two-way ANOVA with Tukey's, n=offspring from 5 SPF, 7 ABX, 4 GF, 5 Sp dams). Data are presented as mean±SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, n.s.=not statistically significant.

FIGS. 4A-4E: L1 thalamocortical axons in embryonic brains of E14.5 offspring from gnotobiotic dams. 4A and 4B, Representative immunofluorescence images of Netrin-G1a (magenta in the original image) and L1 (cyan in the original image) in rostral to caudal embryonic brain sections from E14.5 offspring of SPF, ABX, GF and Sp-colonized dams. Scale bar=500 μm. 4C, Average L1 fluorescence intensity normalized to matched area of region of interest (“yellow-in-the-original-image” dotted lines) across 800 μm of rostral to caudal sections from embryonic brain sections of E14.5 offspring from SPF, ABX, GF and Sp-colonized dams. (Two-way ANOVA with Tukey's, n=offspring from 5 SPF, 7 ABX, 4 GF, 5 Sp dams). 4D, Average area of L1-positive staining across 800 μm of rostral to caudal sections from embryonic brain sections of E14.5 offspring from SPF, ABX, GF and Sp-colonized dams. (Two-way ANOVA with Tukey's, n=offspring from 4 SPF, 5 ABX, 3 GF, 5 Sp dams). 4E, L1 mean fluorescence intensity per L1-positive area across 800 μm of rostral to caudal sections from embryonic brain sections of E14.5 offspring from SPF, ABX, GF and Sp-colonized dams. (Two-way ANOVA with Tukey's, n=offspring from 4 SPF, 4 ABX, 4 GF, 4 Sp dams). Data are presented as mean±SEM, *p<0.05, **p<0.01, ****p<0.0001, n. s.=not statistically significant.

FIGS. 5A-5L: Number and length of axons from thalamic explant monocultures and co-cultures with striatal and hypothalamic explants. 5A, Diagram of E14.5 thalamic (Th) explant monoculture for the axon outgrowth assay. Representative immunofluorescence image of axon outgrowth from 48-hour monoculture of thalamic explants from E14.5 offspring of SPF, ABX and Sp-colonized dams. Scale bar=250 μm. 5B, Number of axons in monoculture of Th explants from E14.5 offspring of SPF, ABX and Sp-colonized dams (One-way ANOVA with Tukey's, n=offspring from 26 SPF, 20 ABX, 10 Sp dams). 5C, Length of axons in SPF, ABX, and Sp Th explant monoculture (One-way ANOVA with Tukey's, n=offspring from 20 dams). 5D, Schematic of E14.5 Th, striatal (St) and hypothalamic (Hy) explant co-culture in axon outgrowth assay. The bar (gray colored in the original image; to the right of Th) indicates site of Th axon quantification, proximal to Hy. Representative fluorescence images of axon outgrowth from i) SPF Th explant proximal to SPF Hy explant (top left), ii) ABX Th explant proximal to ABX Hy explant (top right), iii) SPF Th explant proximal to ABX Hy explant (bottom left), iv) ABX Th explant proximal to SPF Hy explant (bottom right). Scale bar=250 μm. 5E, Number of axons per 200 μm of Th perimeter proximal to Hy explant, normalized to measurements from corresponding Th monoculture. (One-way ANOVA with Tukey's, n=offspring from 14 SPF+SPF, 20 ABX+ABX, 9 SPF+ABX, 10 ABX+SPF dams). 5F, Length of Th axons proximal to Hy explant, normalized to measurements from corresponding Th monoculture. (One-way ANOVA with Tukey's, n=offspring from 14 SPF+SPF, 18 ABX+ABX, 9 SPF+ABX, 10 ABX+SPF dams). 5G, Schematic of E14.5 Th, St and Hy explant co-culture for axon outgrowth assay. The bar (gray colored in the original image; to the left of Th) indicates site of Th axon quantification, proximal to St. Representative fluorescence images of axon outgrowth from a i) SPF Th explant proximal to SPF St explant (top left), ii) ABX Th explant proximal to ABX St explant (top right), iii) Sp Th explant proximal to Sp St explant (bottom left), iv) Sp Th explant proximal to ABX St explant (bottom right). Scale bar=250 μm. 5H, Number of axons per 200 μm of Th perimeter proximal to St explant, normalized to a measurement from corresponding Th monoculture. (One-way ANOVA with Tukey's, n=offspring from 14 SPF+SPF, 20 ABX+ABX, 14 Sp+Sp, 6 Sp+ABX dams). 5I, Length of Th axons proximal to ST explant, normalized to measurements from corresponding Th monoculture. (One-way ANOVA with Tukey's, n=offspring from 14 SPF+SPF, 18 ABX+ABX, 14 Sp+Sp, 6 Sp+ABX dams). 5J, Schematic of E14.5 Th, St and Hy explant co-culture in axon outgrowth assay. The bar (gray colored in the original image; to the right of Th) indicates site of Th axon quantification, proximal to Hy. Representative fluorescence images of axon outgrowth from a i) SPF Th explant proximal to an SPF Hy explant (top left), ii) ABX Th explant proximal to an ABX Hy explant (top right), iii) Sp Th explant proximal to a Sp Hy explant (bottom left), iv) Sp Th explant proximal to an ABX Hy explant (bottom right). Scale bar=250 μm. Red (in the original image) arrows highlight sparse short axons. 5K, Number of axons per 200 μm of Th perimeter proximal to Hy explant, normalized to a measurement from Th corresponding monoculture. (One-way ANOVA with Tukey's, n=offspring from 14 SPF+SPF, 18 ABX+ABX, 14 Sp+Sp, 6 Sp+ABX dams). 5L, Length of Th axons proximal to Hy explant, normalized to measurements from corresponding Th monoculture. (One-way ANOVA with Tukey's, n=offspring from 14 SPF+SPF, 18 ABX+ABX, 14 Sp+Sp, 6 Sp+ABX dams). Data are presented as mean±SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, n. s.=not statistically significant.

FIGS. 6A-6G: Depletion of the maternal microbiota during early gestation yields adult offspring with deficient tactile sensory behavior. 6A, Experimental timeline of vehicle or ABX treatment at 1 week prior to timed mating, conventionalization with SPF bedding on E14.5, and offspring behavioral testing at 6-8 weeks. 6B, The von Frey filament test applies filaments with increasing force (0.4, 0.6, 1, 1.4, 2, 4 grams) to the hindpaw to identify the threshold mechanical force needed to elicit a sensorimotor response. 6C, Force filament required to induce 50% paw withdrawal in adult offspring of SPF and ABX dams (One-way ANOVA with Tukey's, n=offspring from 5 SPF and 7 ABX dams). 6D, Adhesive removal test for sensorimotor behavioral measures sensitivity to detect and dexterity to remove an adhesive tape placed on the mouse forepaw. 6E, Forepaw sensitivity, as measured as the latency to contact the adhesive tape, in adult offspring of SPF and ABX dams (One-way ANOVA with Tukey's, n=offspring from 6 dams per group). 6F, Forepaw motor dexterity, as measured as the latency to remove the adhesive tape after first contact, in adult offspring of SPF and ABX dams (One-way ANOVA with Tukey's, n=offspring from 6 dams per group). 6G, Data for latency to contact and latency to remove the forepaw adhesive in individual mice. (Two-way ANOVA with Sidak's, n=offspring from 6 dams per group). Data are presented as mean±SEM. Data shown for SPF and ABX are as displayed in FIGS. 10A-10M, 12A-12I, 14A-14I, 7A-7F, and 17A-17F. Statistics reflect analysis together with experimental groups: Sp. **p<0.01, ***p<0.001.

FIGS. 7A-7F: Absence of sex differences in behavioral performance of offspring from gnotobiotic dams. 7A, Force filament required to induce 50% paw withdrawal in adult offspring of SPF, ABX, and Sp dams (One-way ANOVA with Tukey's, n=10 SPF, 23 ABX, 25 Sp offspring). 7B, Latency to contact the adhesive tape, in adult offspring of SPF, ABX, Sp dams (One-way ANOVA with Tukey's, n=25 SPF, 19 ABX, 39 Sp offspring). 7C, Latency to remove the adhesive tape after first contact in adult offspring of SPF, ABX, and Sp dams (One-way ANOVA with Tukey's, n=25 SPF, 19 ABX, 39 Sp offspring). 7D, Force filament required to induce 50% paw withdrawal in male and female adult offspring of SPF, ABX, and Sp dams. Male and female comparisons per litter (left) or individual offspring (right) (Two-way ANOVA with Tukey's, n=10 male, 10 female SPF; 7 male, 16 female ABX; 13 male, 12 female Sp offspring). 7E, Latency to contact the adhesive tape in male and female adult offspring of SPF, ABX, Sp dams. Male and female comparisons per litter (left) or individual offspring (right) (Two-way ANOVA with Tukey's, 15 male, 10 female SPF; 4 male, 15 female ABX; 21 male, 18 female Sp offspring). 7F, Latency to remove the adhesive tape after first contact in male and female adult offspring of SPF, ABX, and Sp dams. Male and female comparisons per litter (left) or individual offspring (right) (Two-way ANOVA with Tukey's, n=15 male, 10 female SPF; 4 male, 15 female ABX; 20 male, 18 female Sp offspring). Data are presented as mean±SEM. *p<0.05, **p<0.01, ***p<0.001, n.s=not statistically significant.

FIGS. 8A-8F: Thermal, visual, motor and acoustic sensory behaviors in adult offspring of gnotobiotic dams. 8A, Latency to withdraw in response to a hot plate in adult offspring of SPF, ABX, and Sp-colonized dams (One-way ANOVA with Tukey's; n=9 SPF, 19 ABX, 25 Sp offspring). 8B, Visual depth discrimination determined by a response towards the “safe” or “cliff” zone in the visual cliff test in adult offspring of SPF, ABX, and Sp-colonized dams (One-way ANOVA with Tukey's; n=9 SPF, 19 ABX, 25 Sp offspring). 8C, Percentage of time spent investigating novel texture in whisker texture test in adult offspring of SPF, ABX, and Sp-colonized dams (One-way ANOVA with Tukey's; n=5 SPF, 15 ABX, 18 Sp offspring). 8D, Time spent on the rotarod in adult offspring of SPF, ABX, and Sp-colonized dams (One-way ANOVA with Tukey's; n=45 SPF, 20 ABX, 55 Sp offspring). 8E, Habituation in response to three trials of 120 db acoustic tone in adult offspring of SPF, ABX, and Sp-colonized mice (Two-way ANOVA with Tukey's; n=45 SPF, 15 ABX, 54 Sp offspring). 8F, Inhibitory effect of 70, 75, and 80 db prepulse on startle response to a 120 db acoustic tone in adult offspring of SPF, ABX, and Sp-colonized mice (Two-way ANOVA with Tukey's; n=45 SPF, 15 ABX, 54 Sp offspring). Data are presented as mean±SEM. *p<0.05, **p<0.01, n. s=not statistically significant.

FIGS. 9A-9F: Fetal brain gene expression in offspring of dams colonized with a consortium of spore-forming bacteria (Sp). 9A, Principal coordinate analysis of 16S rDNA sequencing data of feces from SPF compared to Sp-colonized dams at E14.5 (n=4 dams per group) 9B, Taxonomic diversity of the fecal microbiota of SPF and Sp-colonized dams (n=4 dams per group). 9C, Heatmap of genes that were similarly differentially expressed in embryonic brains from E14.5 offspring of SPF and SP dams compared to ABX dams (p<0.05; Wald test, n=offspring from 3 dams per group). Data shown for SPF and ABX are as displayed in FIGS. 1A-1M. Red font (in the original image) indicates axonogenesis-related genes tested by qRT-PCR. 9D, qRT-PCR for PRR12 expression in embryonic brains from E14.5 offspring of SPF, ABX, and Sp-colonized dams (One-way ANOVA with Tukey's; n=offspring from 11 SPF, 15 ABX, 8 Sp dams). 9E, qRT-PCR for NRCAM, NRP2, PLXNA3, and SEMA3F expression in embryonic brains from E14.5 offspring of SPF, ABX, and Sp-colonized dams (Two-way ANOVA with Tukey's; n=offspring from 10 SPF, 10 ABX, 8 Sp dams). 9F, qRT-PCR for expression of axonogenesis-related genes NTN1, SLIT1, DCC, UNC5A, UNC5B, UNC5C, ROBO1, ROBO2 in embryonic brains from E14.5 offspring of SPF, ABX, and Sp-colonized dams (Two-way ANOVA with Tukey's; n=offspring from 11 SPF, 16 ABX, 8 Sp dams). Data are presented as mean±SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, n. s.=not statistically significant.

FIGS. 10A-10M: Gnotobiotic colonization of the maternal microbiota during early gestation prevents neurodevelopmental and behavioral abnormalities induced by maternal microbiota depletion. 10A, Heatmap of differentially expressed genes (p<0.05) in embryonic brains from E14.5 offspring of SPF, ABX, and Sp-colonized dams (Wald test, n=offspring from 3 dams). 10B, Expression of NTNG1 and LRRC4C as measured by quantitative RT-PCR in embryonic brains of E14.5 offspring from SPF, ABX, and Sp-colonized dams (Two-way ANOVA with Tukey's, n=offspring from 9 SPF, 15 ABX, 8 Sp dams). 10C, Representative immunofluorescence images of Netrin-G1a (magenta in the original image) and L1 (cyan in the original image) staining in embryonic brain sections (rostral to caudal) from E14.5 offspring from different dams. Scale bar=500 μm. 10D, Average Netrin-G1a fluorescence intensity per matched area of region of interest (ROI) (“yellow-in-the-original-image” dotted lines) across 800 μm of rostral to caudal embryonic brain sections of E14.5 offspring from SPF, ABX, and Sp-colonized dams. (Two-way ANOVA with Tukey's, n=offspring from 5 SPF, 7 ABX, 5 Sp dams). 10E, Average L1 fluorescence intensity of L1 per matched area of ROI (“yellow-in-the-original-image” dotted lines) across 800 μm of rostral to caudal embryonic brain sections of E14.5 offspring from SPF, ABX, and Sp-colonized dams (Two-way ANOVA with Tukey's, n=offspring from 5 SPF, 7 ABX, 5 Sp dams). 10F, Representative 3-D rendering of Netrin-G1a staining (green in the original image) in cleared whole embryonic brains from E14.5 offspring of Sp dams. Scale bar=100 μm. 10G, Volume of Netrin-G1a axons in cleared whole embryonic brains from E14.5 offspring of SPF, ABX, and Sp-colonized dams. (One-way ANOVA with Tukey's; n=offspring from 5 dams per group). 10H, Length of Netrin-G1a axons in cleared whole embryonic brains from E14.5 offspring of SPF, ABX, and GF dams. (One-way ANOVA with Tukey's; n=offspring from 5 dams per group). 10I, Distance from distal tip of Netrin-G1a staining to the cortex in whole embryonic brains from E14.5 offspring of SPF, ABX, and Sp-colonized dams. (One-way ANOVA with Tukey's; n=offspring from 5 dams per group). 10J, Average circumference of the Netrin-G1a axonal bundle at the internal capsule (IC) in whole embryonic brains from E14.5 offspring of SPF, ABX, and Sp-colonized dams. (One-way ANOVA with Tukey's; n=offspring from 5 dams per group). 10K, Experimental timeline of ABX treatment and Sp colonization prior to breeding, conventionalization with SPF microbiota on E14.5, and offspring behavioral testing at 6-8 weeks. 10L, Force filament required to induce 50% paw withdrawal in adult offspring of SPF, ABX and Sp-colonized dams (One-way ANOVA with Tukey's, n=offspring from 5 SPF, 7 ABX, 7 Sp dams). 10M, left: Forepaw sensitivity, as measured as the latency to contact the adhesive tape, in adult offspring of SPF, ABX and Sp-colonized dams (One-way ANOVA with Tukey's, n=offspring from 6 SPF, 6 ABX, 7 Sp dams). right: Forepaw motor dexterity, as measured as the latency to remove the adhesive tape after first contact, in adult offspring of SPF, ABX and Sp-colonized dams (One-way ANOVA with Tukey's, n=offspring from 6 SPF, 6 ABX, 7 Sp dams). Data are presented as mean±SEM. Data shown for SPF and ABX are as displayed in FIGS. 1A-1M, 6A-6G, 14A-14I, 3A-3I, 4A-4D, 5A-5L, and 7A-7F. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, n. s.=not statistically significant.

FIGS. 11A-11D: Fetal Netrin-G1a thalamocortical axons offspring of dams colonized with a consortium of Bacteroides species (BD). 11A, Taxonomic diversity of the fecal microbiota of SPF and BD colonized dams (n=6 dams). 11B, Representative immunofluorescence images of Netrin-G1a (magenta in the original image) and L1 (cyan in the original image) staining in embryonic brain sections (rostra′ to caudal) from E14.5 offspring of BD-colonized dams. Scale bar=500 μm. 11C, Average Netrin-G1a fluorescence intensity per matched area of region of interest (ROI) (“yellow-in-the-original-image” dotted lines) across 800 μm of rostral to caudal embryonic brain sections of E14.5 offspring from SPF, ABX, and BD-colonized dams. (Two-way ANOVA with Tukey's, n=offspring from 5 SPF, 7 ABX, 4 BD dams). 11D, Average L1 fluorescence intensity per matched area of ROI (“yellow-in-the-original-image” dotted lines) across 800 μm of rostral to caudal embryonic brain sections of E14.5 offspring from SPF, ABX and BD-colonized dams. (Two-way ANOVA with Tukey's, n=offspring from 5 SPF, 7 ABX, 4 BD dams). Data are presented as mean±SEM. Data shown for SPF and ABX are as displayed in FIGS. 1A-1M, 10A-10M, 2A-2D, 3A-I. *p<0.05, **p<0.01, ****p<0.0001, n. s.=not statistically significant.

FIGS. 12A-12I: The maternal microbiota modulates fetal brain metabolites during pregnancy. 12A, Unsupervised hierarchical clustering of 753 maternal serum metabolites, classified by metabolite super pathway (n=6 dams per group). 12B, Principal component analysis of 753 metabolites detected in maternal serum metabolomes from SPF, ABX, GF, and Sp-colonized dams on E14.5 (n=6 dams per group) 12C, Principal component analysis of 567 metabolites detected in fetal brain metabolomes from E14.5 offspring of SPF, ABX, GF, and Sp-colonized dams (n=offspring from 6 dams per group). 12D, Volcano plot of significantly regulated metabolites in SPF vs ABX (left) and SPF v GF (right). Orange dots (in the original image) indicate log 2fold>0.5. Red dots (in the original image) indicate p-value<0.05. Green dots (in the original image) indicate metabolites that are log 2fold>0.5 and p-value<0.05 (ANOVA with contrasts, n=offspring from 6 dams per group). 12E, Volcano plot of significantly regulated metabolites in Sp vs ABX (left) and Sp v GF (right). Orange dots (in the original image) indicate log 2fold>0.5. Red dots (in the original image) indicate p-value<0.05. Green dots (in the original image) indicate metabolites that are log 2fold>0.5 and p-value<0.05 (ANOVA with contrasts, n=offspring from 6 dams per group). 12F, Amino acid, lipid and xenobiotic metabolites significantly dysregulated in fetal brains of E14.5 offspring from ABX-treated dams compared to SPF controls (p<0.05; One-Way ANOVA, n=offspring from 6 dams per group). 12G, Amino acid, lipid and xenobiotic metabolites significantly dysregulated in fetal brains of E14.5 offspring from ABX-treated dams compared to Sp controls (p<0.05; One-Way ANOVA, n=offspring from 6 dams per group). 12H, Random Forest classification of top 30 metabolites in fetal brain that discriminate between maternal colonization with SPF and Sp microbiota compared to ABX and GF. Red highlight (in the original image) indicates select metabolites with >2-fold decrease in ABX and GF compared to SPF and Sp (n=offspring from 6 dams per group). 12I, Select metabolites significantly decreased in fetal brains from E14.5 embryos of ABX and GF dams versus SPF and Sp dams. (One-way ANOVA with Tukey's, n=offspring from 6 dams per group). Data are presented as mean±SEM. **p<0.01, ***p<0.001, ****p<0.0001.

FIGS. 13A-13C: The maternal microbiota modulates maternal serum metabolites during pregnancy. 13A, Random Forest classification of top 30 metabolites in maternal serum that discriminate between maternal colonization with SPF and Sp compared to ABX and GF. Red font (in the original image) indicates metabolites similarly regulated in both maternal serum and fetal brain from SPF and Sp compared to ABX and GF conditions (n=6 dams per group). 13B, Relative concentrations of N, N, N-trimethyl-5-aminovalerate, trimethylamine N-oxide, imidazole propionate, hippurate, and 3-indoxyl-sulfate in maternal sera of SPF, ABX, GF, and Sp dams (One-way ANOVA with Tukey's; n=6 dams per group). 13C, Amino acid, lipid and xenobiotic metabolites significantly dysregulated in fetal brains of E14.5 offspring from GF dams compared to SPF controls (left) and GF dams compared to Sp dams (right) (p<0.05; One-way ANOVA, n=offspring from 6 dams per group). Data are presented as mean±SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, n. s.=not statistically significant.

FIGS. 14A-14I: The maternal microbiota modulates metabolites that promote fetal thalamocortical axonogenesis and adult sensory behavior. 14A, Schematic of axon outgrowth assay with individual metabolite supplementation. 14B, Representative fluorescence images of axon outgrowth from ABX thalamic explants proximal to an ABX striatal explant (top left), and supplemented with i) vehicle (top left), ii) Trimethylamine N-oxide (TMAO, 1 nM) iii) 5-aminovalerate (5-AV, 1 nM), iv) imidazole propionate (IP, 1 nM), v) 3-indoxyl-sulfate (3-IS, 1 nM) or vi) Hippurate (HIP, 1 nM). Scale bar=250 μm. 14C, Number of axons per 200 μm of thalamic perimeter proximal to striatal explant (One-way ANOVA with Tukey's, n=offspring from 14 SPF+SPF, 13 ABX+ABX, 7 TMAO, 7 5-AV, 7 IP, 7 3-IS, 8 HIP dams). 14D, Representative immunofluorescence images of Netrin-G1a (magenta in the original image) and L1 (cyan in the original image) staining in embryonic brain sections (rostral to caudal) from E14.5 offspring of ABX dams treated with vehicle or 4-microbial metabolites (4-MM: TMAO, 5-AV, IP, and HIP). Scale bar=500 μm. 14E, Average Netrin-G1a fluorescence intensity per matched area of region of interest (ROI) (“yellow-in-the-original-image” dotted lines) across 800 μm of rostral to caudal embryonic brain sections of E14.5 offspring from ABX dams treated with vehicle or 4-MM. (Two-way ANOVA with Tukey's, n=offspring from 8 dams per group). 14F, Average L1 fluorescence intensity of L1 per matched area of ROI (“yellow-in-the-original-image” dotted lines) across 800 μm of rostral to caudal embryonic brain sections of E14.5 offspring from ABX dams treated with vehicle or 4-MM. (Two-way ANOVA with Tukey's, n=offspring from 8 dams per group). 14G, Experimental timeline for ABX+Veh and ABX+4-MM groups. 14H, Force filament to induce 50% paw withdrawal in adult offspring of SPF, ABX, ABX+Veh, and ABX+4-MM dams (One-way ANOVA with Tukey's, n=offspring from 5 SPF, 7 ABX, 6 ABX+Veh, 7 ABX+4-MM dams). 14I, Left: Latency to contact the adhesive tape in adult offspring of SPF, ABX, ABX+Veh and ABX+4-MM dams (One-way ANOVA with Tukey's, n=offspring from 6 SPF, 6 ABX, 5 ABX+Veh, 5 ABX+4-MM dams). Middle: Latency to remove the adhesive tape after first contact in offspring of SPF, ABX, ABX+Veh and ABX+4-MM dams (One-way ANOVA with Tukey's, n=offspring from 6 SPF, 6 ABX, 5 ABX+Veh, 5 ABX+4-MM dams). Right: Pairwise comparison of latency to contact and latency to remove in offspring of SPF, ABX, ABX+Veh and ABX+4-MM dams (Two-way ANOVA with Sidak's, n=offspring from 6 SPF, 6 ABX, 5 ABX+Veh, 5 ABX+4-MM dams). Data shown for SPF and ABX as reference controls are as presented in FIGS. 1A-1M. 6A-6G, and 10A-10M. Data are presented as mean±SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, n. s.=not statistically significant.

FIGS. 15A-15D: Dose effects of microbiome-dependent metabolites on thalamocortical axon outgrowth. 15A, Number of axons per 200 μm of thalamic perimeter proximal to striatal explant from i) SPF thalamic explant proximal to an SPF striatal explant (“SPF+SPF St”, left), as positive control ii) ABX thalamic explant proximal to an ABX striatal explant (“ABX+ABX St”), as negative control, and iii) ABX+ABX St, supplemented with 1 nM, 100 nM, 10 uM of metabolites: trimethylamine N-oxide (TMAO), 5-aminovalerate (5-AV), imidazole propionate (IP), 3-indoxyl-sulfate (3-IS) or Hippurate (HIP). (One-way ANOVA with Tukey's, n=offspring from 14 SPF+SPF; 13 ABX+ABX; 7, 6, 7 TMAO; 3, 5, 7 5-AV; 5, 7, 7 IP; 3, 7, 7 3-IS; 6, 7, 8 HIP dams). 15B, Length of axons per 200 μm of thalamic perimeter proximal to striatal explant from i) SPF+SPF St as positive control ii) ABX+ABX St, as negative control, and iii) ABX+ABX St, supplemented with 1 nM, 100 nM, 10 1.1M of metabolites: TMAO, 5-AV, IP, 3-IS or HIP. (One-way ANOVA with Tukey's, n=offspring from 14 SPF+SPF; 12 ABX+ABX; 7, 6, 6 TMAO; 3, 5, 6 5-AV; 5, 7, 6 IP; 3, 7, 4 3-IS; 6, 7, 7 HIP dams). 15C, Number of axons per 200 μm of thalamic perimeter proximal to hypothalamic explant (Hy) from i) SPF+SPF Hy, as positive control ii) ABX+ABX Hy as negative control, and iii) ABX+ABX Hy, supplemented with 1 nM, 100 nM, 10 uM of metabolites: TMAO, 5-AV, IP, 3-IS or HIP. (One-way ANOVA with Tukey's, n=offspring from 14 SPF+SPF; 10 ABX+ABX; 6, 6, 7 TMAO; 3, 5, 7 5-AV; 3, 6, 7 IP; 3, 7, 5 3-IS; 5, 7, 8 HIP dams). 15D, Length of axons per 200 μm of thalamic perimeter proximal to hypothalamic explant from i) SPF+SPF Hy as positive control ii) ABX+ABX Hy as negative control, and iii) ABX+ABX Hy, supplemented with 1 nM, 100 nM, 10 uM of metabolites: TMAO, 5-AV, IP, 3-IS or HIP. (One-way ANOVA with Tukey's, n=offspring from 14 SPF+SPF; 9 ABX+ABX; 6, 6, 6 TMAO; 3, 5, 6 5-AV; 3, 7, 6 IP; 3, 7, 4 3-IS; 5, 7, 7 HIP dams). Data are presented as mean±SEM. *p<0.05. **p<0.01, ***p<0.001, ****p<0.0001, n. s.=not statistically significant.

FIGS. 16A-16D: Netrin-G1a thalamocortical axons in embryonic brains of E14.5 offspring from metabolite supplementation dams. 16A, Immunofluorescence images of Netrin-G1a in four independent embryonic brain sections from E14.5 offspring of ABX+vehicle and ABX+4-MM dams at 200 μm intervals. Scale bar: 500 μm. 16B, Total Netrin-G1a fluorescence intensity across 800 μm of rostral to caudal sections of E14.5 embryonic brains from ABX+vehicle and ABX+4-MM dams. (Two-way ANOVA with Tukey's, n=offspring from 8 dams per group). 16C, Average area of Netrin-G1a-positive staining across 800 μm of rostral to caudal embryonic brain sections of E14.5 offspring from ABX+vehicle and ABX+4-MM dams. (Two-way ANOVA with Tukey's, n=offspring from 8 dams per group). 16D, Average DAPI fluorescence intensity normalized to per matched area of region of interest (“yellow-in-the-original-image” dotted lines) in 800 μm of rostral to caudal embryonic brain sections of E14.5 offspring from ABX+vehicle and ABX+4-MM dams. (Two-way ANOVA with Tukey's, n=offspring from 8 dams per group). Data are presented as mean±SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, n. s.=not statistically significant.

FIGS. 17A-17F: Absence of sex differences in behavioral performance of offspring from metabolite-treated dams. 17A, Force filament required to induce 50% paw withdrawal in adult offspring of SPF, ABX, ABX+Veh, ABX+4-MM dams (One-way ANOVA with Tukey's, n=10 SPF, 23 ABX, 19 ABX+Veh, 29 ABX+4-MM offspring). 17B, Latency to contact the adhesive tape, in adult offspring of SPF, ABX, ABX+Veh, ABX+4-MM dams (One-way ANOVA with Tukey's, n=25 SPF, 19 ABX, 19 ABX+Veh, 29 ABX+4-MM offspring). 17C, Latency to remove the adhesive tape after first contact in adult offspring of SPF, ABX, and Sp dams (One-way ANOVA with Tukey's, n=25 SPF, 19 ABX, 19 ABX+Veh, 29 ABX+4-MM offspring). 17D, Force filament to induce 50% paw withdrawal in male and female adult offspring of SPF, ABX, ABX+Veh, and ABX+4-MM dams. Male and female comparisons per litter (left) or individual offspring (right). (Two-way ANOVA with Tukey's, n=10 male, 10 female SPF; 7 male, 16 female ABX; 9 male, 10 female ABX+Veh; 13 male, 16 female ABX+4-MM dams). 17E, Latency to contact the adhesive tape, in male and female adult offspring of SPF, ABX, ABX+Veh, ABX+4-MM dams. Male and female comparisons per litter (left) or individual offspring (right). (Two-way ANOVA with Tukey's, n=15 male, 10 female SPF; 4 male, 15 female ABX; 6 male, 11 female ABX+Veh; 11 male, 16 female ABX+4-MM dams). 17F, Latency to remove the adhesive tape after first contact in male and female adult offspring of SPF, ABX, and Sp dams. Male and female comparisons per litter (left) or individual offspring (right). (Two-way ANOVA with Tukey's, n=15 male, 10 female SPF; 4 male, 15 female ABX; 6 male, 11 female ABX+Veh; 11 male, 16 female ABX+4-MM dams). Data are presented as mean±SEM. n. s=not statistically significant.

FIG. 18: A schematic depicting that the maternal microbiome mediates brain development and behaviours.

DETAILED DESCRIPTION

In some embodiments, the methods of the present disclosure are directed to promoting healthy neural development in an unborn baby, for example by administering to a subject (e.g., a maternal subject gestating the unborn baby, a female subject who plans to, expects to, or suspects of being pregnant) a composition, a bacterial composition, or both as disclosed herein.

In some embodiments, the methods of the present disclosure are directed to methods of conditioning a female subject for fostering healthy neural development in offspring, for example by administering to the subject a composition, a bacterial composition, or both as disclosed herein.

A composition that can be administered in these methods may comprise trimethylamine N-oxide (TMAO), 5-aminovalerate (5-AV), imidazole propionate (IP), hippurate (HIP), or a combination thereof (e.g., 5-AV and IP; 5-AV, IP, and TMAO; all 4 (4-MM)).

A bacterial composition that can be administered in these methods may comprise bacteria of the order Clostridiales. These bacteria can be of any of the following families: Lachnospiraceae, Ruminococcaceae, Clostridiaceae, or a combination thereof. In some embodiments, these bacteria are of any of the following genuses: Clostridium, Dehalobacterium, Ruminococcus, Coprococcus, Dorea, Oscillospira, or a combination thereof. In certain embodiments, these bacteria are spore-forming bacteria.

Healthy neural development can include healthy thalamocortical axon growth, healthy netrin-G1a+thalamocortical axogenesis, healthy tactile sensory development, or a combination thereof.

The disclosed compositions can be administered at various times. For example, they can be administered at least once (e.g., once during the full period, twice during the full period, once a day) during a period that runs from the first day of an expected-but-missed menstruation to a day that is two months after said first day. An alternative timing can be a period that runs from the second day of the expected-but-missed menstruation to a day that is 37 days after said second day (e.g., which for humans corresponds approximately to the mouse period from E7.5 to E14.5, which in units of days post conception (dpc) can be from 17 dpc to 52 dpc, at least in some subjects). Such timings can be useful to female subjects who prefer not to or cannot get tested for pregnancy though a professional facility. The administration time can also be at least once during a two-month period that ends with the day of an expected conception for the female subject. Such a timing can be useful for a subject who is planning pregnancy. The timing is, in some embodiments, at least once within the first trimester, second trimester, third trimester, or a combination thereof. More specific periods include the period that runs from the start of the third week after conception to the end of the eighth week after conception, and the period that runs from the 17^(th) dpc to the 52^(nd) dpc.

The disclosed methods can also be used to reduce adverse effects of antibiotic treatment on an unborn baby in a pregnant subject. For example, administering to the pregnant subject a composition that comprises trimethylamine N-oxide (TMAO), 5-aminovalerate (5-AV), imidazole propionate (IP), hippurate (HIP), or a combination thereof can promote healthy neural development, at least in comparison to a lack of such administration, in the unborn baby.

In certain embodiments, the methods of the present disclosure are directed to methods for selecting a female subject for conditioning to foster healthy neural development in offspring. These methods include determining that a compound has a level in a serum sample from the female subject that is at most 10%, 20%, 30%, 40%, 50%, 60%, or 70% of its level in a control serum sample representative of a healthy female subject, that bacteria of the order Clostridiales have a total level in a fecal sample from the female subject that is at most 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 14%, 16%, 18%, or 20% of their total level in a control fecal sample representative of a healthy female subject, or both, and selecting the female subject for conditioning to foster healthy neural development in offspring. In some embodiments, these criteria can be relaxed. For example, even if a subject has levels (of the compound, of the bacteria) that are similar to those of a healthy control, the subject may still be selected for treatment (e.g., with the bacterial compositions, which can be part of the normal gastrointestinal microbiome of a human) as a prophylactic measure.

In various embodiments, offspring can include babies carried by a surrogate mother, in which the baby need not be the biological offspring of the gestating female.

In these methods of selecting a female subject, the compound can be 2-(4-hydroxyphenyl)propionate; 3-(3-hydroxyphenyl)propionate sulfate; 3-indoxyl sulfate; 3-phenylpropionate (hydrocinnamate); 7-ketodeoxycholate; alpha-ketoglutaramate; alpha-muricholate; beta-muricholate; biotin; deoxycholate; hippurate; imidazole propionate; indolepropionate; N,N,N-trimethyl-5-aminovalerate; p-cresol sulfate; phenylpropionylglycine; pyrraline; stachydrine; taurodeoxycholate; taurohyodeoxycholic acid; trimethylamine N-oxide; ursodeoxycholate; or a combination thereof. Alternatively, the compound can be 3-indoxyl sulfate; biotin; hippurate; imidazole propionate; N,N,N-trimethyl-5-aminovalerate; pyrraline; stachydrine; trimethylamine N-oxide; or a combination thereof. The bacteria, in some of these embodiments, includes bacteria of the genus Clostridium, genus Dehalobacterium, genus Ruminococcus, genus Coprococcus, genus Dorea, genus Oscillospira, or a combination thereof. Once a female subject is selected, she can be treated by administering to her a composition, bacterial composition, or both as provided herein.

In certain embodiments, the methods of the present disclosure are directed to promoting healthy neural development in a fetus, such as by administering to a maternal subject gestating the fetus (or to a female subject) a composition as described herein. Preferably, the method results in the fetus exhibiting a lesser degree of impaired neural development relative to a fetus gestated by similar a maternal subject (e.g., a maternal subject having a similar or identical maternal microbiome) not receiving the composition. Preferably, the method results in an increase in one or more of fetal brain gene expression, fetal axonogenesis (e.g., fetal thalamocortical axonogenesis), fetal axon development, and adult tactile sensory behavior relative to a fetus gestated by similar a maternal subject (e.g., a maternal subject having a similar or identical maternal microbiome) not receiving the composition.

In additional embodiments of any of the aspects disclosed herein, the conjugate base forms or the conjugate acid forms of the disclosed compounds can be used, either instead of or together with their conjugate form. For example, in certain embodiments, hippuric acid can be used instead of or in addition to hippurate, imidazolepropionic acid can be used instead of or in addition to imidazole propionate, and 5-aminovaleric acid can be used instead of or in addition to 5-aminovalerate.

In certain embodiments, the methods of the present disclosure are directed to inhibiting development of a disease or disorder in a fetus, e.g., by administering to a maternal subject gestating the fetus (or to a female subject) a composition as described herein. Preferably, the method results in the fetus exhibiting a lesser degree of development of the disease or disorder (e.g., a metabolic disorder, a cardiovascular disorder, a cerebrovascular disorder, stroke, Alzheimer's disease, schizophrenia, depression, or autism) during the fetal period and throughout the lifetime of the eventual child, adolescent, and adult, relative to a fetus gestated by a similar maternal subject (e.g., a maternal subject having a similar or identical maternal microbiome) not receiving the composition.

In certain embodiments, the methods further comprise administering the composition to the maternal subject or a female subject prior to gestation. In certain embodiments, the female subject is a fertile, ovulating female subject. In certain embodiments, the female subject is a female subject seeking to implant an embryo.

In certain embodiments, the composition comprises a compound selected from trimethylamine-N-oxide (TMAO), N,N,N-trimethyl-5-aminovaleric acid (TMAV), imidazole propionic acid (IP), 3-indoxyl sulfuric acid (3-IS), hippuric acid (HIP), perfluorooctanesulfonic acid (PFOS), 3-sulfo-L-alanine, alpha-ketoglutaramic acid, 4-hydroxyglutamic acid, extoine, stachydrine, biotin, pyroglutamine, chiro-inositol, N1-methyl-2-pyridone-5-carboxamide, O-sulfo-L-tyrosine, 2′deoxyuridine, pyrraline, N-delta-acetylornithine, phenyl sulfuric acid, phenylacetylglycine, anserine, homostachydrine, serine, N1-methyl-4-pyridone-3-carboxamide, methyl glycopyranoside (alpha+beta), 3-carboxy-1-methylpyridin-1-ium, hypotaurine, 1,5-anhydroglucitol (1,5-AG), 1-oleoyl-2-linoleoyl-GPC (18:1/18:2), methionine, glutamine, malic acid, pantothenic acid, 5,6-dihydrouridine, ceramide (d18:1/17:0 d17:1/18.0) N6-methyllysine, allantoin, N2-acetyllysine, N-acetylglutamine, stearoylcarnitine (C18), arachidoylcarnitine (C20), arabitol, xylitol, 1-methylhistamine, xanthosine, xanthine, 1-ribosyl-imidazoleacetic acid, 5-methyl-2′-deoxycytidine, sphingomyelin (d18:0/20:0, d16:0/22:0), 1-methyl-4-imidazoleacetic acid, inosine 5′-monophosphoric acid (IMP), 1,2-distearoyl-GPC (18:0/18:0), 3-methylcytidine, pipecolate, N-stearoyl-sphingadienine (d18:2/18:0), homoserine, carnosine, 1-palmitoyl-GPI (16:0), 1-stearoyl-GPI (18:0), N6-succinyladenosine, 2′-deoxycytidine, stearoyl-docosahexaenoyl-glycerol (18:0/22:6), trigonelline (N′-methylnicotinate), hydroxyasparagine, gamma-glutamylglutamic acid, 2-palmitoylglycerol (16:0), ceramide (d18:1/17:0, d17:1/18:0), thiamin (Vitamin B1), N6-methyllysine, N6,N6-dimethyllysine, 3-hydroxy-3-methylglutaric acid, campesterol, allantoin, stachydrine, N2-acetyllysine, phenyllactic acid (PLA), gamma-glutamyltryptophan, N-palmitoyl-sphingosine (d18:1/16:0), O-sulfo-L-tyrosine, indolelactic acid, gamma-glutamylglutamine, N-acetylglucosamine 6-phosphoric acid, 1-oleoyl-GPS (18:1), 3-hydroxypalmitoylcarnitine, myo-inositol, behenoyl sphingomyelin (d18:1/22:0), maltotetraose, maltotriose, N-acetylglucosamine/N-acetylgalactosamine, N1-methyladenosine, uracil, 1-oleoyl-GPI (18:1), sphingomyelin (d18:1/17:0, d17:1/18:0, d19:1/16:0), 3-ureidopropionic acid, 5-oxoproline, gamma-glutamyltyrosine, 1-(1-enyl-stearoyl)-GPE (P-18:0), cytidine 2′,3′-cyclic monophosphoric acid, 2′-deoxyguanosine 5′-monophosphoric acid (dGMP), thymidine, N6,N6,N6-trimethyllysine, 1-palmitoyl-GPC (16:0), 1-(1-enyl-palmitoyl)-GPE (P-16:0), N-stearoyl-sphinganine (d18:0/18:0), N-arachidoyl-sphingosine (d18:1/20:0), 3′-dephosphocoenzyme A, 5-hydroxylysine, arabonic acid/xylonic acid, 1-palmitoyl-2-docosahexaenoyl-GPC (16:0/22:6), glutamine, 1-(1-enyl-palmitoyl)-2-palmitoyl-GPC (P-16:0/16:0), N-behenoyl-sphingadienine (d18:2/22:0), xylulose 5-phosphoric acid, 1-oleoyl-GPC (18:1), 1-stearoyl-GPE (18:0), glycerol 3-phosphoric acid, N-stearoyl-sphingosine (d18:1/18:0), 7-methylguanine, N2,N2-dimethylguanosine, N-acetylglutamine, methionine, pro-hydroxy-pro, dihydroxyacetone phosphoric acid (DHAP), 1-stearoyl-2-docosahexaenoyl-GPC (18:0/22:6), sphingomyelin (d18:1/20:0, d16:1/22:0), uric acid, adenylosuccinic acid, cystathionine, spermine, mannitol/sorbitol, 2-hydroxyadipic acid, N-palmitoyl-sphinganine (d18:0/16:0), sphingomyelin (d18:0/18:0, d19:0/17:0), sphingomyelin (d18:1/24:1, d18:2/24:0), alpha-hydroxyisovaleric acid, citrulline, ribulonic acid/xylulonic acid, succinylcarnitine (C4-DC), ceramide (d16:1/24:1, d18:1/22:1), hypoxanthine, 5,6-dihydrouridine, gamma-aminobutyric acid (GABA), oleoyl ethanolamide, choline, 1-palmitoyl-GPE (16:0), palmitoyl-linoleoyl-glycerol (16:0/18:2), ceramide (d18:2/24:1, d18:1/24:2), cholesterol, 2′-O-methylcytidine, nicotinamide riboside, pantothenic acid, pyridoxal, N-acetylaspartic acid (NAA), C-glycosyltryptophan, methionine sulfoxide, spermidine, 1-palmitoyl-2-oleoyl-GPG (16:0/18:1), lignoceroyl sphingomyelin (d18:1/24:0), desmosterol, N1-methylinosine, cytidine, N-acetyl-aspartyl-glutamic acid (NAAG), sedoheptulose, galactonic acid, cytidine 5′-monophospho-N-acetylneuraminic acid, glycerophosphoinositol, uridine, salicylic acid, N-acetylglutamic acid, gamma-glutamyl-epsilon-lysine, glycerophosphoserine, 1-stearoyl-2-oleoyl-GPE (18:0/18:1), beta-alanine, 5-methylcytidine, methylphosphoric acid, imidazole lactic acid, sedoheptulose-7-phosphoric acid, 1-palmitoyl-2-stearoyl-GPE (16:0/18:0), guanosine 5′-diphosphoric acid (GDP), 3-ureidoisobutyric acid, tryptophan, isoleucine, methyl succinic acid, S-adenosylmethionine (SAM), taurine, gamma-glutamylthreonine, arabitol/xylitol, erythronic acid, fumaric acid, stearoylcarnitine (C18), deoxycarnitine, cytidine 5′-diphosphocholine, 1-stearoyl-2-arachidonoyl-GPC (18:0/20:4), 1-stearoyl-2-arachidonoyl-GPI (18:0/20:4), glycerophosphoglycerol, N6-carbamoylthreonyladenosine, flavin adenine dinucleotide (FAD), 2-oxoarginine, lactic acid, gulonic acid, phenylalanine, 3-(4-hydroxyphenyl)lactic acid, 2-hydroxyglutaric acid, palmitoleoyl ethanolamide, 1-palmitoyl-2-stearoyl-GPC (16:0/18:0), 1-stearoyl-2-oleoyl-GPC (18:0/18:1), palmitoyl-oleoyl-glycerol (16:0/18:1), betaine, N-acetylneuraminic acid, malic acid, phosphoethanolamine, 1-myristoyl-2-arachidonoyl-GPC (14:0/20:4), beta-citrylglutamic acid, 1-methylhistidine, leucine, ethylmalonic acid, prolylglycine, stearoyl-arachidonoyl-glycerol (18:0/20:4), orotidine, 5-(galactosylhydroxy)-L-lysine, N-acetylglucosaminylasparagine, eicosenoylcarnitine (C20:1), cytidine-5′-diphosphoethanolamine, glycosyl-N-stearoyl-sphingosine (d18:1/18:0), palmitoyl dihydrosphingomyelin (d18:0/16:0), sphingosine, inosine, guanosine 5′-monophosphoric acid (5′-GMP), dimethylglycine, N-acetylalanine, aspartic acid, creatine, ribitol, 2-methylcitric acid/homocitric acid, arachidoylcarnitine (C20), S-methylglutathione, 1-palmitoyl-2-arachidonoyl-GPC (16:0/20:4n6), stearoyl sphingomyelin (d18:1/18:0), nicotinamide, N-formylmethionine, UDP-N-acetylglucosamine/galactosamine, glucoronic acid, 1,2-dipalmitoyl-GPE (16:0/16:0), pseudouridine, alanine, glutamic acid, 1-myristoyl-2-palmitoyl-GPC (14:0/16:0), 1,2-dipalmitoyl-GPC (16:0/16:0), 1-palmitoyl-2-oleoyl-GPC (16:0/18:1), sphingomyelin (d18:1/22:1, d18:2/22:0, d16:1/24:1), glycerophosphoethanolamine, 1-palmitoyl-2-palmitoleoyl-GPC (16:0/16:1), UDP-glucuronic acid, and 1-methylnicotinamide, or a salt thereof, or a combination thereof.

In certain embodiments, the composition comprises a compound selected from trimethylamine-N-oxide (TMAO), N,N,N-trimethyl-5-aminovaleric acid (TMAV), imidazole propionic acid (IP), 3-indoxyl sulfuric acid (3-IS), hippuric acid (HIP), perfluorooctanesulfonic acid (PFOS), 3-sulfo-L-alanine, alpha-ketoglutaramic acid, 4-hydroxyglutamic acid, extoine, stachydrine, biotin, pyroglutamine, chiro-inositol, N1-methyl-2-pyridone-5-carboxamide, O-sulfo-L-tyrosine, 2′deoxyuridine, pyrraline, N-delta-acetylornithine, phenylsulfuric acid, phenylacetylglycine, anserine, homostachydrine, serine, N1-methyl-4-pyridone-3-carboxamide, methyl glycopyranoside (alpha+beta), 3-carboxy-1-methylpyridin-1-ium, hypotaurine, 1,5-anhydroglucitol (1,5-AG), 1-oleoyl-2-linoleoyl-GPC (18:1/18:2), methionine, glutamine, malic acid, pantothenic acid, 5,6-dihydrouridine, ceramide (d18:1/17:0 d17:1/18.0) N6-methyllysine, allantoin, N2-acetyllysine, N-acetylglutamine, stearoylcarnitine (C18), arachidoylcarnitine (C20), arabitol, and xylitol, or a salt thereof, or a combination thereof. In certain embodiments, the composition comprises a compound selected from 3-sulfo-L-alanine, TMAV, IP, TMAO, 3-IS, phenylsulfuric acid, stachydrine, hippuric acid, homostachydrine, pyrraline, alpha-ketoglutaramic acid, O-sulfo-L-tyrosine, methionine, 3-carboxy-1-methylpyridin-1-ium, biotin, glutamine, malic acid, pantothenic acid, pyroglutamine, anserine, 5,6-dihydrouridine, phenylacetylglycine, ceramide (d18:1/17:0 d17:1/18.0), N6-methyllysine, allantoin, N2-acetyllysine, N-acetylglutamine, stearoylcarnitine (C18), arachidoylcarnitine (C20), arabitol, and xylitol, or a salt thereof, or a combination thereof. In certain embodiments, the composition comprises a compound selected from methionine, glutamine, malic acid, pantothenic acid, 5,6-dihydrouridine, ceramide (d18:1/17:0 d17:1/18.0), N6-methyllysine, allantoin, N2-acetyllysine, N-acetylglutamine, stearoylcarnitine (C18), arachidoylcarnitine (C20), arabitol, and xylitol, or a salt thereof, or a combination thereof. In certain embodiments, the composition comprises a compound selected from TMAO, TMAV, HIP, IP, and 3-IS, or a salt thereof, or a combination thereof. In certain embodiments, the composition comprises a compound selected from TMAO, TMAV, and HIP, or a salt thereof, or a combination thereof. In certain embodiments, the composition comprises a compound selected from TMAO, TMAV, IP, and 3-IS, or a salt thereof, or a combination thereof. In certain embodiments, the composition comprises a compound selected from TMAO, TMAV, IP, and HIP, or a salt thereof, or a combination thereof. In certain embodiments, the composition comprises a compound selected from TMAV or TMAO, or a salt thereof, or a combination thereof. In certain embodiments, the composition comprises the compound TMAO or a salt thereof.

In some embodiments, the present invention is drawn to a composition comprising at least one bacterial species or bacterial strain (e.g., a probiotic bacterial strain) capable of promoting healthy neural development in a fetus and/or inhibiting development of a disease or disorder in a subject, optionally wherein the at least one bacterial species or bacterial strain is alive and capable of proliferation. Such bacteria (e.g., probiotic bacteria) inhibit one or more adverse effects of maternal microbiota depletion (e.g., in ABX subjects) on neural development, e.g., fetal brain gene expression, thalamocortical axon outgrowth, and offspring sensory behavior. In some embodiments, such bacteria restore expression of one or more genes relevant to axon guidance. In certain embodiments, the at least one bacterial species or bacterial strain is a bacterial species found in a maternal microbiome. In some embodiments, the one or more bacterial species is a spore-forming bacterial species.

In certain embodiments, the one or more bacteria in the composition are spore-forming bacteria. In certain embodiments, the one or more spore-forming bacteria are selected from order Clostridiales (e.g., family Lachnospiraceae, family Clostridiaceae, family Ruminococcaceae, family Dehalobacteriaceae), order Turicibacterales (e.g., family Turicibacteraceae), order Anaeroplasmatales (e.g., family Anaeroplasmataceae), order Erysipelotrichales (e.g., family Erysipelotrichaceae), and order RF39, or a combination thereof. In certain embodiments, the one or more spore-forming bacteria are selected from order Clostridiales.

In certain embodiments, the one or more bacteria in the composition are selected from order Lactobacillales (e.g., genus Enterococcus), order Clostridiales (e.g., family Clostridiaceae, family Peptostreptococcaceae, family Lachnospiraceae), order Turicibacterales (e.g., genus Turicibacter), order Erysipelotrichales (e.g., genus Eubacterium), order Enterobacteriales, order Bacteroidales (e.g., genus Bacteroides), or a combination thereof. In certain embodiments, the one or more bacteria are selected from order Bacteroidales (e.g., genus Bacteroides).

In certain embodiments, the one or more bacteria in the composition are selected from phylum Firmicutes, phylum Tenericutes, phylum Bacteroidetes, or a combination thereof. In certain embodiments, the one or more bacteria in the composition from phylum Firmicutes comprises one or more bacteria selected from class Clostridia, class Bacilli (e.g., order Lactobacillales, order Turicibacterales), class Erysipelotrichi, and class Gammaproteobacteria. In certain embodiments, the one or more bacteria in the composition from phylum Bacteroidetes comprises one or more bacteria selected from genus Bacteroides (e.g., B. thetraiotaomicron, B. uniformis, B. vulgatus, B. ovatus, B. fragilus). In certain embodiments, the one or more bacteria in the composition from phylum Tenericutes comprises one or more bacteria selected from class Mollicutes (e.g., order Anaeroplasmatales, order RF39).

Definitions

“Impaired neural development,” as used herein, refers to abnormalities in brain function and behavior, in offspring. Examples of impaired neural development include, but are not limited to, impairments in fetal brain gene expression, fetal axonogenesis (such as fetal thalamocortical axonogenesis), and/or adult tactile sensory behavior (e.g., tactile hyposensitivity in sensorimotor behavioral tasks). Examples of “healthy neural development,” as used herein, include, but are not limited to, healthy development in fetal brain gene expression, fetal axonogenesis, fetal axon development, and/or adult tactile sensory behavior.

“Microbiome,” as used herein, refers to the microorganisms in a given environment, such as the body or a part of the body. The “maternal microbiome,” as used herein, refers to the microorganisms in a maternal subject (i.e., a pregnant or gestating subject), particularly in the gut of the maternal subject. The gut microbiome modulates the bioavailability of hundreds of biochemicals in the circulating blood. During pregnancy, the maternal gut environment supplies nutrients and growth factors, from the maternal diet and other nutritional intake, to nurture offspring growth.

A “depleted” maternal microbiome is characterized by a reduced level of one or more microbial species (e.g., one or more bacterial species), such as 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of the level relative to a maternal subject without a depleted maternal microbiome.

“Germ-free” (GF) subjects, as used herein, are subjects with no microorganisms living in or on them. “Antibiotic-treated” (ABX) subjects, as used herein, are subjects treated with one or more antibiotic compounds, many representative examples of which are known in the art.

The term “subject” to which administration is contemplated includes, but is not limited to, humans (i.e., a male or female of any age group, e.g., a pediatric subject (e.g., infant, child, adolescent) or adult subject (e.g., young adult, middle-aged adult or senior adult)) and/or other primates (e.g., cynomolgus monkeys, rhesus monkeys); and/or mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, goats, cats, and/or dogs. Preferred subjects are humans.

An “ovulating” female subject, as used herein, refers to a female subject having a regular cycle of menses, e.g., a female between menarche and menopause that is not employing hormonal birth control that inhibits ovulation. A “fertile” female subject, as used herein, refers to an ovulating female subject able to conceive offspring.

As used herein, a therapeutic that “prevents” a disorder or condition refers to a compound or composition that, in a statistical sample, reduces the occurrence of the disorder or condition in the treated sample relative to an untreated control sample, or delays the onset or reduces the severity of one or more symptoms of the disorder or condition relative to the untreated control sample.

The term “treating” includes prophylactic and/or therapeutic treatments. The term “prophylactic or therapeutic” treatment is art-recognized and includes administration to the subject of one or more of the disclosed compositions. If it is administered prior to clinical manifestation of the unwanted condition (e.g., disease or other unwanted state of the subject) then the treatment is prophylactic (i.e., it protects the subject against developing the unwanted condition), whereas if it is administered after manifestation of the unwanted condition, the treatment is therapeutic (i.e., it is intended to diminish, ameliorate, or stabilize the existing unwanted condition or side effects thereof).

The term “prodrug” is intended to encompass compounds which, under physiologic conditions, are converted into therapeutically active agents. A common method for making a prodrug is to include one or more selected moieties which are hydrolyzed under physiologic conditions to reveal the desired molecule. In other embodiments, the prodrug is converted by an enzymatic activity of the host animal. For example, esters or carbonates (e.g., esters or carbonates of alcohols or carboxylic acids) and esters or amides of phosphates and phosphonic acids are preferred prodrugs of the present invention.

As used herein, the term “about” is defined as being close to as understood by one of ordinary skill in the art. In one non-limiting embodiment, the term “about” is defined to be within 10%, preferably within 5%, more preferably within 1%, and most preferably within 0.5%.

As used herein, “stably stored” or “storage-stable” refer to a composition in which cells are able to withstand storage for extended periods of time (e.g., at least one month, or two, three, four, six, or twelve months or more) with a less than 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15%, 10%, 5%, or 1% decrease in cell viability.

As used herein, the phrase “conjoint administration” refers to any form of administration of two or more different therapeutic compounds such that the second compound is administered while the previously administered therapeutic compound is still effective in the body (e.g., the two compounds are simultaneously effective in the subject, which may include synergistic effects of the two compounds). For example, the different therapeutic compounds can be administered either in the same formulation or in a separate formulation, either concomitantly or sequentially. In certain embodiments, the different therapeutic compounds can be administered within one hour, 12 hours, 24 hours, 36 hours, 48 hours, 72 hours, or a week of one another. Thus, a subject who receives such treatment can benefit from a combined effect of different therapeutic compounds.

Bacterial Compositions

In certain aspects, provided herein are bacterial compositions that include bacteria of the order Clostridiales. In some embodiments, the bacteria of the order Clostridiales include bacteria of the family Lachnospiraceae, family Ruminococcaceae, family Clostridiaceae, or a combination thereof. In certain embodiments, the bacteria of the order Clostridiales include bacteria of the genus Clostridium, genus Dehalobacterium, genus Ruminococcus, genus Coprococcus, genus Dorea, genus Oscillospira, or a combination thereof. The bacteria of the order Clostridiales can be spore-forming bacteria. In some embodiments, the bacteria are selected from those presented in Table 2.

In certain aspects, provided herein are bacterial compositions comprising one or more bacteria and optionally a compound selected from trimethylamine-N-oxide (TMAO), N,N,N-trimethyl-5-aminovaleric acid (TMAV), imidazole propionic acid (IP), 3-indoxyl sulfuric acid (3-IS), hippuric acid (HIP), perfluorooctanesulfonic acid (PFOS), 3-sulfo-L-alanine, alpha-ketoglutaramic acid, 4-hydroxyglutamic acid, extoine, stachydrine, biotin, pyroglutamine, chiro-inositol, N1-methyl-2-pyridone-5-carboxamide, O-sulfo-L-tyrosine, 2′deoxyuridine, pyrraline, N-delta-acetylornithine, phenylsulfuric acid, phenylacetylglycine, anserine, homostachydrine, serine, N1-methyl-4-pyridone-3-carboxamide, methyl glycopyranoside (alpha+beta), 3-carboxy-1-methylpyridin-1-ium, hypotaurine, 1,5-anhydroglucitol (1,5-AG), 1-oleoyl-2-linoleoyl-GPC (18:1/18:2), methionine, glutamine, malic acid, pantothenic acid, 5,6-dihydrouridine, ceramide (d18:1/17:0 d17:1/18.0) N6-methyllysine, allantoin, N2-acetyllysine, N-acetylglutamine, stearoylcarnitine (C18), arachidoylcarnitine (C20), arabitol, xylitol, 1-methylhistamine, xanthosine, xanthine, 1-ribosyl-imidazoleacetic acid, 5-methyl-2′-deoxycytidine, sphingomyelin (d18:0/20:0, d16:0/22:0), 1-methyl-4-imidazoleacetic acid, inosine 5′-monophosphoric acid (IMP), 1,2-distearoyl-GPC (18:0/18:0), 3-methylcytidine, pipecolate, N-stearoyl-sphingadienine (d18:2/18:0), homoserine, carnosine, 1-palmitoyl-GPI (16:0), 1-stearoyl-GPI (18:0), N6-succinyladenosine, 2′-deoxycytidine, stearoyl-docosahexaenoyl-glycerol (18:0/22:6), trigonelline (N′-methylnicotinate), hydroxyasparagine, gamma-glutamylglutamic acid, 2-palmitoylglycerol (16:0), ceramide (d18:1/17:0, d17:1/18:0), thiamin (Vitamin B1), N6-methyllysine, N6,N6-dimethyllysine, 3-hydroxy-3-methylglutaric acid, campesterol, allantoin, stachydrine, N2-acetyllysine, phenyllactic acid (PLA), gamma-glutamyltryptophan, N-palmitoyl-sphingosine (d18:1/16:0), O-sulfo-L-tyrosine, indolelactic acid, gamma-glutamylglutamine, N-acetylglucosamine 6-phosphoric acid, 1-oleoyl-GPS (18:1), 3-hydroxypalmitoylcarnitine, myo-inositol, behenoyl sphingomyelin (d18:1/22:0), maltotetraose, maltotriose, N-acetylglucosamine/N-acetylgalactosamine, N1-methyladenosine, uracil, 1-oleoyl-GPI (18:1), sphingomyelin (d18:1/17:0, d17:1/18:0, d19:1/16:0), 3-ureidopropionic acid, 5-oxoproline, gamma-glutamyltyrosine, 1-(1-enyl-stearoyl)-GPE (P-18:0), cytidine 2′,3′-cyclic monophosphoric acid, 2′-deoxyguanosine 5′-monophosphoric acid (dGMP), thymidine, N6,N6,N6-trimethyllysine, 1-palmitoyl-GPC (16:0), 1-(1-enyl-palmitoyl)-GPE (P-16:0), N-stearoyl-sphinganine (d18:0/18:0), N-arachidoyl-sphingosine (d18:1/20:0), 3′-dephosphocoenzyme A, 5-hydroxylysine, arabonic acid/xylonic acid, 1-palmitoyl-2-docosahexaenoyl-GPC (16:0/22:6), glutamine, 1-(1-enyl-palmitoyl)-2-palmitoyl-GPC (P-16:0/16:0), N-behenoyl-sphingadienine (d18:2/22:0), xylulose 5-phosphoric acid, 1-oleoyl-GPC (18:1), 1-stearoyl-GPE (18:0), glycerol 3-phosphoric acid, N-stearoyl-sphingosine (d18:1/18:0), 7-methylguanine, N2,N2-dimethylguanosine, N-acetylglutamine, methionine, pro-hydroxy-pro, dihydroxyacetone phosphoric acid (DHAP), 1-stearoyl-2-docosahexaenoyl-GPC (18:0/22:6), sphingomyelin (d18:1/20:0, d16:1/22:0), uric acid, adenylosuccinic acid, cystathionine, spermine, mannitol/sorbitol, 2-hydroxyadipic acid, N-palmitoyl-sphinganine (d18:0/16:0), sphingomyelin (d18:0/18:0, d19:0/17:0), sphingomyelin (d18:1/24:1, d18:2/24:0), alpha-hydroxyisovaleric acid, citrulline, ribulonic acid/xylulonic acid, succinylcarnitine (C4-DC), ceramide (d16:1/24:1, d18:1/22:1), hypoxanthine, 5,6-dihydrouridine, gamma-aminobutyric acid (GABA), oleoyl ethanolamide, choline, 1-palmitoyl-GPE (16:0), palmitoyl-linoleoyl-glycerol (16:0/18:2), ceramide (d18:2/24:1, d18:1/24:2), cholesterol, 2′-O-methylcytidine, nicotinamide riboside, pantothenic acid, pyridoxal, N-acetylaspartic acid (NAA), C-glycosyltryptophan, methionine sulfoxide, spermidine, 1-palmitoyl-2-oleoyl-GPG (16:0/18:1), lignoceroyl sphingomyelin (d18:1/24:0), desmosterol, N1-methylinosine, cytidine, N-acetyl-aspartyl-glutamic acid (NAAG), sedoheptulose, galactonic acid, cytidine 5′-monophospho-N-acetylneuraminic acid, glycerophosphoinositol, uridine, salicylic acid, N-acetylglutamic acid, gamma-glutamyl-epsilon-lysine, glycerophosphoserine, 1-stearoyl-2-oleoyl-GPE (18:0/18:1), beta-alanine, 5-methylcytidine, methylphosphoric acid, imidazole lactic acid, sedoheptulose-7-phosphoric acid, 1-palmitoyl-2-stearoyl-GPE (16:0/18:0), guanosine 5′-diphosphoric acid (GDP), 3-ureidoisobutyric acid, tryptophan, isoleucine, methyl succinic acid, S-adenosylmethionine (SAM), taurine, gamma-glutamylthreonine, arabitol/xylitol, erythronic acid, fumaric acid, stearoylcarnitine (C18), deoxycarnitine, cytidine 5′-diphosphocholine, 1-stearoyl-2-arachidonoyl-GPC (18:0/20:4), 1-stearoyl-2-arachidonoyl-GPI (18:0/20:4), glycerophosphoglycerol, N6-carbamoylthreonyladenosine, flavin adenine dinucleotide (FAD), 2-oxoarginine, lactic acid, gulonic acid, phenylalanine, 3-(4-hydroxyphenyl)lactic acid, 2-hydroxyglutaric acid, palmitoleoyl ethanolamide, 1-palmitoyl-2-stearoyl-GPC (16:0/18:0), 1-stearoyl-2-oleoyl-GPC (18:0/18:1), palmitoyl-oleoyl-glycerol (16:0/18:1), betaine, N-acetylneuraminic acid, malic acid, phosphoethanolamine, 1-myristoyl-2-arachidonoyl-GPC (14:0/20:4), beta-citrylglutamic acid, 1-methylhistidine, leucine, ethylmalonic acid, prolylglycine, stearoyl-arachidonoyl-glycerol (18:0/20:4), orotidine, 5-(galactosylhydroxy)-L-lysine, N-acetylglucosaminylasparagine, eicosenoylcarnitine (C20:1), cytidine-5′-diphosphoethanolamine, glycosyl-N-stearoyl-sphingosine (d18:1/18:0), palmitoyl dihydrosphingomyelin (d18:0/16:0), sphingosine, inosine, guanosine 5′-monophosphoric acid (5′-GMP), dimethylglycine, N-acetylalanine, aspartic acid, creatine, ribitol, 2-methylcitric acid/homocitric acid, arachidoylcarnitine (C20), S-methylglutathione, 1-palmitoyl-2-arachidonoyl-GPC (16:0/20:4n6), stearoyl sphingomyelin (d18:1/18:0), nicotinamide, N-formylmethionine, UDP-N-acetylglucosamine/galactosamine, glucoronic acid, 1,2-dipalmitoyl-GPE (16:0/16:0), pseudouridine, alanine, glutamic acid, 1-myristoyl-2-palmitoyl-GPC (14:0/16:0), 1,2-dipalmitoyl-GPC (16:0/16:0), 1-palmitoyl-2-oleoyl-GPC (16:0/18:1), sphingomyelin (d18:1/22:1, d18:2/22:0, d16:1/24:1), glycerophosphoethanolamine, 1-palmitoyl-2-palmitoleoyl-GPC (16:0/16:1), UDP-glucuronic acid, and 1-methylnicotinamide, or a salt thereof, or a combination thereof.

In certain aspects, provided herein are bacterial compositions comprising one or more bacteria and optionally a compound selected from trimethylamine-N-oxide (TMAO), N,N,N-trimethyl-5-aminovaleric acid (TMAV), imidazole propionic acid (IP), 3-indoxyl sulfuric acid (3-IS), hippuric acid (HIP), perfluorooctanesulfonic acid (PFOS), 3-sulfo-L-alanine, alpha-ketoglutaramic acid, 4-hydroxyglutamic acid, extoine, stachydrine, biotin, pyroglutamine, chiro-inositol, N1-methyl-2-pyridone-5-carboxamide, O-sulfo-L-tyrosine, 2′deoxyuridine, pyrraline, N-delta-acetylornithine, phenylsulfuric acid, phenylacetylglycine, anserine, homostachydrine, serine, N1-methyl-4-pyridone-3-carboxamide, methyl glycopyranoside (alpha+beta), 3-carboxy-1-methylpyridin-1-ium, hypotaurine, 1,5-anhydroglucitol (1,5-AG), 1-oleoyl-2-linoleoyl-GPC (18:1/18:2), methionine, glutamine, malic acid, pantothenic acid, 5,6-dihydrouridine, ceramide (d18:1/17:0 d17:1/18.0) N6-methyllysine, allantoin, N2-acetyllysine, N-acetylglutamine, stearoylcarnitine (C18), arachidoylcarnitine (C20), arabitol, and xylitol, or a salt thereof, or a combination thereof. In certain aspects, provided herein are bacterial compositions comprising one or more bacteria and optionally a compound selected from 3-sulfo-L-alanine, TMAV, IP, TMAO, 3-IS, phenylsulfuric acid, stachydrine, hippuric acid, homostachydrine, pyrraline, alpha-ketoglutaramic acid, O-sulfo-L-tyrosine, methionine, 3-carboxy-1-methylpyridin-1-ium, biotin, glutamine, malic acid, pantothenic acid, pyroglutamine, anserine, 5,6-dihydrouridine, phenylacetylglycine, ceramide (d18:1/17:0 d17:1/18.0), N6-methyllysine, allantoin, N2-acetyllysine, N-acetylglutamine, stearoylcarnitine (C18), arachidoylcarnitine (C20), arabitol, and xylitol, or a salt thereof, or a combination thereof. In certain embodiments, provided herein are bacterial compositions comprising one or more bacteria and optionally a compound selected from methionine, glutamine, malic acid, pantothenic acid, 5,6-dihydrouridine, ceramide (d18:1/17:0 d17:1/18.0), N6-methyllysine, allantoin, N2-acetyllysine, N-acetylglutamine, stearoylcarnitine (C18), arachidoylcarnitine (C20), arabitol, and xylitol, or a salt thereof, or a combination thereof. In certain embodiments, provided herein are bacterial compositions comprising one or more bacteria and optionally a compound selected from TMAO, TMAV, HIP, IP, and 3-IS, or a salt thereof, or a combination thereof. In certain embodiments, provided herein are bacterial compositions comprising one or more bacteria and optionally a compound selected from TMAO, TMAV, and HIP, or a salt thereof, or a combination thereof. In certain embodiments, provided herein are bacterial compositions comprising one or more bacteria and optionally a compound selected from TMAO, TMAV, IP, and 3-IS, or a salt thereof, or a combination thereof. In certain embodiments, provided herein are bacterial compositions comprising one or more bacteria and optionally a compound selected from TMAO, TMAV, IP, and HIP, or a salt thereof, or a combination thereof. In certain embodiments, provided herein are bacterial compositions comprising one or more bacteria and optionally a compound selected from TMAV or TMAO, or a salt thereof, or a combination thereof. In certain embodiments, provided herein are bacterial compositions comprising one or more bacteria and optionally TMAO or a salt thereof. In certain embodiments, the one or more bacteria in the composition are spore-forming bacteria.

Preferably the bacterium is of a bacterial species found in the maternal microbiome (e.g., the maternal gut microbiome), including, but not limited to, a bacterial species selected from spore-forming bacteria (such as order Clostridiales (e.g., family Lachnospiraceae, family Clostridiaceae, family Ruminococcaceae, family Dehalobacteriaceae), order Turicibacterales (e.g., family Turicibacteraceae), order Anaeroplasmatales (e.g., family Anaeroplasmataceae), order Erysipelotrichales (e.g., family Erysipelotrichaceae), and order RF39), order Lactobacillales (e.g., genus Enterococcus), order Clostridiales (e.g., family Clostridiaceae, family Peptostreptococcaceae, family Lachnospiraceae), order Turicibacterales (e.g., genus Turicibacter), order Erysipelotrichales (e.g., genus Eubacterium), order Enterobacteriales, order Bacteroidales (e.g., genus Bacteroides), phylum Firmicutes (e.g., class Clostridia, class Bacilli (e.g., order Lactobacillales, order Turicibacterales), class Erysipelotrichi, and class Gammaproteobacteria), phylum Bacteroidetes (e.g., genus Bacteroides (such as B. thetraiotaomicron, B. uniformis, B. vulgatus, B. ovatus, B. fragilus)), phylum Tenericutes (e.g., class Mollicutes (e.g., order Anaeroplasmatales, order RF39)), or a combination thereof. In some embodiments, the bacterial formulation comprises a bacterium and/or a combination of bacteria described herein and a pharmaceutically acceptable carrier.

In certain embodiments, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the bacteria in the bacterial composition are spore-forming bacteria selected from order Clostridiales (e.g., family Lachnospiraceae, family Clostridiaceae, family Ruminococcaceae, family Dehalobacteriaceae), order Turicibacterales (e.g., family Turicibacteraceae), order Anaeroplasmatales (e.g., family Anaeroplasmataceae), order Erysipelotrichales (e.g., family Erysipelotrichaceae), and order RF39, or a combination thereof, such as order Clostridiales. In certain embodiments, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the bacteria in the bacterial composition are selected from order Lactobacillales (e.g., genus Enterococcus), order Clostridiales (e.g., family Clostridiaceae, family Peptostreptococcaceae, family Lachnospiraceae), order Turicibacterales (e.g., genus Turicibacter), order Erysipelotrichales (e.g., genus Eubacterium), order Enterobacteriales, and order Bacteroidales (e.g., genus Bacteroides), or a combination thereof. In certain embodiments, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the bacteria in the bacterial composition are selected from phylum Firmicutes, phylum Tenericutes, phylum Bacteroidetes, or a combination thereof.

In certain embodiments, substantially all of the bacteria in the bacterial composition are spore-forming bacteria selected from order Clostridiales (e.g., family Lachnospiraceae, family Clostridiaceae, family Ruminococcaceae, family Dehalobacteriaceae), order Turicibacterales (e.g., family Turicibacteraceae), order Anaeroplasmatales (e.g., family Anaeroplasmataceae), order Erysipelotrichales (e.g., family Erysipelotrichaceae), and order RF39, or a combination thereof, such as order Clostridiales. In certain embodiments, substantially all of the bacteria in the bacterial composition are selected from order Lactobacillales (e.g., genus Enterococcus), order Clostridiales (e.g., family Clostridiaceae, family Peptostreptococcaceae, family Lachnospiraceae), order Turicibacterales (e.g., genus Turicibacter), order Erysipelotrichales (e.g., genus Eubacterium), order Enterobacteriales, and order Bacteroidales (e.g., genus Bacteroides), or a combination thereof. In certain embodiments, substantially all of the bacteria in the bacterial composition are phylum Firmicutes, phylum Tenericutes, or phylum Bacteroidetes, or a combination thereof.

In certain embodiments, the bacterial composition comprises at least 1×10³ colony forming units (CFUs), 1×10⁴ colony forming units (CFUs), 1×10⁵ colony forming units (CFUs), 5×10⁵ colony forming units (CFUs), 1×10⁶ colony forming units (CFUs), 2×10⁶ colony forming units (CFUs), 3×10⁶ colony forming units (CFUs), 4×10⁶ colony forming units (CFUs), 5×10⁶ colony forming units (CFUs), 6×10⁶ colony forming units (CFUs), 7×10⁶ colony forming units (CFUs), 8×10⁶ colony forming units (CFUs), 9×10⁶ colony forming units (CFUs), 1×10⁷ colony forming units (CFUs), 2×10⁷ colony forming units (CFUs), 3×10⁷ colony forming units (CFUs), 4×10⁷ colony forming units (CFUs), 5×10⁷ colony forming units (CFUs), 6×10⁷ colony forming units (CFUs), 7×10⁷ colony forming units (CFUs), 8×10⁷ colony forming units (CFUs), 9×10⁷ colony forming units (CFUs), 1×10⁸ colony forming units (CFUs), 2×10⁸ colony forming units (CFUs), 3×10⁸ colony forming units (CFUs), 4×10⁸ colony forming units (CFUs), 5×10⁸ colony forming units (CFUs), 6×10⁸ colony forming units (CFUs), 7×10⁸ colony forming units (CFUs), 8×10⁸ colony forming units (CFUs), 9×10⁸ colony forming units (CFUs), 1×10⁹ colony forming units (CFUs), 5×10⁹ colony forming units (CFUs), 1×10¹⁰ colony forming units (CFUs) 5×10¹⁰ colony forming units (CFUs), 1×10¹¹ colony forming units (CFUs) 5×10¹¹ colony forming units (CFUs), 1×10¹² colony forming units (CFUs) 5×10¹² colony forming units (CFUs), 1×10¹³ colony forming units (CFUs) spore-forming bacteria selected from order Clostridiales (e.g., family Lachnospiraceae, family Clostridiaceae, family Ruminococcaceae, family Dehalobacteriaceae), order Turicibacterales (e.g., family Turicibacteraceae), order Anaeroplasmatales (e.g., family Anaeroplasmataceae), order Erysipelotrichales (e.g., family Erysipelotrichaceae), and order RF39, or a combination thereof, such as order Clostridiales.

In certain embodiments, the bacterial composition comprises at least 1×10³ colony forming units (CFUs), 1×10⁴ colony forming units (CFUs), 1×10⁵ colony forming units (CFUs), 5×10⁵ colony forming units (CFUs), 1×10⁶ colony forming units (CFUs), 2×10⁶ colony forming units (CFUs), 3×10⁶ colony forming units (CFUs), 4×10⁶ colony forming units (CFUs), 5×10⁶ colony forming units (CFUs), 6×10⁶ colony forming units (CFUs), 7×10⁶ colony forming units (CFUs), 8×10⁶ colony forming units (CFUs), 9×10⁶ colony forming units (CFUs), 1×10⁷ colony forming units (CFUs), 2×10⁷ colony forming units (CFUs), 3×10⁷ colony forming units (CFUs), 4×10⁷ colony forming units (CFUs), 5×10⁷ colony forming units (CFUs), 6×10⁷ colony forming units (CFUs), 7×10⁷ colony forming units (CFUs), 8×10⁷ colony forming units (CFUs), 9×10⁷ colony forming units (CFUs), 1×10⁸ colony forming units (CFUs), 2×10⁸ colony forming units (CFUs), 3×10⁸ colony forming units (CFUs), 4×10⁸ colony forming units (CFUs), 5×10⁸ colony forming units (CFUs), 6×10⁸ colony forming units (CFUs), 7×10⁸ colony forming units (CFUs), 8×10⁸ colony forming units (CFUs), 9×10⁸ colony forming units (CFUs), 1×10⁹ colony forming units (CFUs), 5×10⁹ colony forming units (CFUs), 1×10¹⁰ colony forming units (CFUs) 5×10¹⁰ colony forming units (CFUs), 1×10¹¹ colony forming units (CFUs) 5×10¹¹ colony forming units (CFUs), 1×10¹² colony forming units (CFUs) 5×10¹² colony forming units (CFUs), 1×10¹³ colony forming units (CFUs) of bacteria selected from order Lactobacillales (e.g., genus Enterococcus), order Clostridiales (e.g., family Clostridiaceae, family Peptostreptococcaceae, family Lachnospiraceae), order Turicibacterales (e.g., genus Turicibacter), order Erysipelotrichales (e.g., genus Eubacterium), order Enterobacteriales, and order Bacteroidales (e.g., genus Bacteroides), or a combination thereof.

In certain embodiments, the bacterial composition comprises at least 1×10³ colony forming units (CFUs), 1×10⁴ colony forming units (CFUs), 1×10⁵ colony forming units (CFUs), 5×10⁵ colony forming units (CFUs), 1×10⁶ colony forming units (CFUs), 2×10⁶ colony forming units (CFUs), 3×10⁶ colony forming units (CFUs), 4×10⁶ colony forming units (CFUs), 5×10⁶ colony forming units (CFUs), 6×10⁶ colony forming units (CFUs), 7×10⁶ colony forming units (CFUs), 8×10⁶ colony forming units (CFUs), 9×10⁶ colony forming units (CFUs), 1×10⁴ colony forming units (CFUs), 2×10⁷ colony forming units (CFUs), 3×10⁷ colony forming units (CFUs), 4×10⁷ colony forming units (CFUs), 5×10⁷ colony forming units (CFUs), 6×10⁷ colony forming units (CFUs), 7×10⁷ colony forming units (CFUs), 8×10⁷ colony forming units (CFUs), 9×10⁷ colony forming units (CFUs), 1×10⁸ colony forming units (CFUs), 2×10⁸ colony forming units (CFUs), 3×10⁸ colony forming units (CFUs), 4×10⁸ colony forming units (CFUs), 5×10⁸ colony forming units (CFUs), 6×10⁸ colony forming units (CFUs), 7×10⁸ colony forming units (CFUs), 8×10⁸ colony forming units (CFUs), 9×10⁸ colony forming units (CFUs), 1×10⁹ colony forming units (CFUs), 5×10⁹ colony forming units (CFUs), 1×10¹⁰ colony forming units (CFUs) 5×10¹⁰ colony forming units (CFUs), 1×10¹¹ colony forming units (CFUs) 5×10¹¹ colony forming units (CFUs), 1×10¹² colony forming units (CFUs) 5×10¹² colony forming units (CFUs), 1×10¹³ colony forming units (CFUs) of phylum Firmicutes, phylum Tenericutes, or phylum Bacteroidetes, or a combination thereof.

The selected dosage level will depend upon a variety of factors including the subject's diet, the route of administration, the time of administration, the residence time of the particular microorganism being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular composition employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could prescribe and/or administer doses of the bacteria employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

In some embodiments, probiotic formulations containing a bacteria selected from spore-forming bacteria (order Clostridiales (e.g., family Lachnospiraceae, family Clostridiaceae, family Ruminococcaceae, family Dehalobacteriaceae), order Turicibacterales (e.g., family Turicibacteraceae), order Anaeroplasmatales (e.g., family Anaeroplasmataceae), order Erysipelotrichales (e.g., family Erysipelotrichaceae), order RF39), order Lactobacillales (e.g., genus Enterococcus), order Clostridiales (e.g., family Clostridiaceae, family Peptostreptococcaceae, family Lachnospiraceae), order Turicibacterales (e.g., genus Turicibacter), order Erysipelotrichales (e.g., genus Eubacterium), order Enterobacteriales, order Bacteroidales (e.g., genus Bacteroides), phylum Firmicutes (e.g., class Clostridia, class Bacilli (e.g., order Lactobacillales, order Turicibacterales), class Erysipelotrichi, and class Gammaproteobacteria), phylum Bacteroidetes (e.g., genus Bacteroides (such as B. thetraiotaomicron, B. uniformis, B. vulgatus, B. ovatus, B. fragilus)), and phylum Tenericutes (e.g., class Mollicutes (e.g., order Anaeroplasmatales, order RF39)), or a combination thereof are provided as encapsulated, enteric coated, or powder forms, with doses ranging up to 10¹¹ cfu (e.g., up to 10¹⁰ cfu). In some embodiments, the composition comprises 5×10¹¹ cfu of a bacteria selected from spore-forming bacteria (order Clostridiales (e.g., family Lachnospiraceae, family Clostridiaceae, family Ruminococcaceae, family Dehalobacteriaceae), order Turicibacterales (e.g., family Turicibacteraceae), order Anaeroplasmatales (e.g., family Anaeroplasmataceae), order Erysipelotrichales (e.g., family Erysipelotrichaceae), order RF39), order Lactobacillales (e.g., genus Enterococcus), order Clostridiales (e.g., family Clostridiaceae, family Peptostreptococcaceae, family Lachnospiraceae), order Turicibacterales (e.g., genus Turicibacter), order Erysipelotrichales (e.g., genus Eubacterium), order Enterobacteriales, order Bacteroidales (e.g., genus Bacteroides), phylum Firmicutes (e.g., class Clostridia, class Bacilli (e.g., order Lactobacillales, order Turicibacterales), class Erysipelotrichi, and class Gammaproteobacteria), phylum Bacteroidetes (e.g., genus Bacteroides (such as B. thetraiotaomicron, B. uniformis, B. vulgatus, B. ovatus, B. fragilus)), and phylum Tenericutes (e.g., class Mollicutes (e.g., order Anaeroplasmatales, order RF39)), or a combination thereof, and 10% (w/w) corn starch in a capsule. In some embodiments, the capsule is enteric coated, e.g., for duodenal release at pH 5.5. In some embodiments, the composition comprises a powder of freeze-dried a bacteria selected from spore-forming bacteria (order Clostridiales (e.g., family Lachnospiraceae, family Clostridiaceae, family Ruminococcaceae, family Dehalobacteriaceae), order Turicibacterales (e.g., family Turicibacteraceae), order Anaeroplasmatales (e.g., family Anaeroplasmataceae), order Erysipelotrichales (e.g., family Erysipelotrichaceae), order RF39), order Lactobacillales (e.g., genus Enterococcus), order Clostridiales (e.g., family Clostridiaceae, family Peptostreptococcaceae, family Lachnospiraceae), order Turicibacterales (e.g., genus Turicibacter), order Erysipelotrichales (e.g., genus Eubacterium), order Enterobacteriales, order Bacteroidales (e.g., genus Bacteroides), phylum Firmicutes (e.g., class Clostridia, class Bacilli (e.g., order Lactobacillales, order Turicibacterales), class Erysipelotrichi, and class Gammaproteobacteria), phylum Bacteroidetes (e.g., genus Bacteroides (such as B. thetraiotaomicron, B. uniformis, B. vulgatus, B. ovatus, B. fragilus)), and phylum Tenericutes (e.g., class Mollicutes (e.g., order Anaeroplasmatales, order RF39)), or a combination thereof, which is deemed to have “Qualified Presumption of Safety” (QPS) status. In some embodiments, the composition is storage-stable at frozen or refrigerated temperature.

Methods for producing microbial compositions may include three main processing steps. The steps are: organism banking, organism production, and preservation. In certain embodiments, a sample that contains an abundance of a bacteria selected from spore-forming bacteria (order Clostridiales (e.g., family Lachnospiraceae, family Clostridiaceae, family Ruminococcaceae, family Dehalobacteriaceae), order Turicibacterales (e.g., family Turicibacteraceae), order Anaeroplasmatales (e.g., family Anaeroplasmataceae), order Erysipelotrichales (e.g., family Erysipelotrichaceae), order RF39), order Lactobacillales (e.g., genus Enterococcus), order Clostridiales (e.g., family Clostridiaceae, family Peptostreptococcaceae, family Lachnospiraceae), order Turicibacterales (e.g., genus Turicibacter), order Erysipelotrichales (e.g., genus Eubacterium), order Enterobacteriales, order Bacteroidales (e.g., genus Bacteroides), phylum Firmicutes (e.g., class Clostridia, class Bacilli (e.g., order Lactobacillales, order Turicibacterales), class Erysipelotrichi, and class Gammaproteobacteria), phylum Bacteroidetes (e.g., genus Bacteroides (such as B. thetraiotaomicron, B. uniformis, B. vulgatus, B. ovatus, B. fragilus)), phylum Tenericutes (e.g., class Mollicutes (e.g., order Anaeroplasmatales, order RF39)), or a combination thereof, may be cultured by avoiding an isolation step.

For banking, a bacteria selected from spore-forming bacteria (order Clostridiales (e.g., family Lachnospiraceae, family Clostridiaceae, family Ruminococcaceae, family Dehalobacteriaceae), order Turicibacterales (e.g., family Turicibacteraceae), order Anaeroplasmatales (e.g., family Anaeroplasmataceae), order Erysipelotrichales (e.g., family Erysipelotrichaceae), order RF39), order Lactobacillales (e.g., genus Enterococcus), order Clostridiales (e.g., family Clostridiaceae, family Peptostreptococcaceae, family Lachnospiraceae), order Turicibacterales (e.g., genus Turicibacter), order Erysipelotrichales (e.g., genus Eubacterium), order Enterobacteriales, order Bacteroidales (e.g., genus Bacteroides), phylum Firmicutes (e.g., class Clostridia, class Bacilli (e.g., order Lactobacillales, order Turicibacterales), class Erysipelotrichi, and class Gammaproteobacteria), phylum Bacteroidetes (e.g., genus Bacteroides (such as B. thetraiotaomicron, B. uniformis, B. vulgatus, B. ovatus, B. fragilus)), and phylum Tenericutes (e.g., class Mollicutes (e.g., order Anaeroplasmatales, order RF39)), or a combination thereof, included in the microbial composition may be (1) isolated directly from a specimen or taken from a banked stock, (2) optionally cultured on a nutrient agar or broth that supports growth to generate viable biomass, and (3) the biomass optionally preserved in multiple aliquots in long-term storage.

In embodiments using a culturing step, the agar or broth may contain nutrients that provide essential elements and specific factors that enable growth. An example would be a medium composed of 20 g/L glucose, 10 g/L yeast extract, 10 g/L soy peptone, 2 g/L citric acid, 1.5 g/L sodium phosphate monobasic, 100 mg/L ferric ammonium citrate, 80 mg/L magnesium sulfate, 10 mg/L hemin chloride, 2 mg/L calcium chloride, 1 mg/L menadione. Another example would be a medium composed of 10 g/L beef extract, 10 g/L peptone, 5 g/L sodium chloride, 5 g/L dextrose, 3 g/L yeast extract, 3 g/L sodium acetate, 1 g/L soluble starch, and 0.5 g/L L-cysteine HCl, at pH 6.8. A variety of microbiological media and variations are well known in the art (e.g., R. M. Atlas, Handbook of Microbiological Media (2010) CRC Press). Culture media can be added to the culture at the start, may be added during the culture, or may be intermittently/continuously flowed through the culture. The strains in the bacterial composition may be cultivated alone, as a subset of the microbial composition, or as an entire collection comprising the microbial composition. As an example, a first strain may be cultivated together with a second strain in a mixed continuous culture, at a dilution rate lower than the maximum growth rate of either cell to prevent the culture from washing out of the cultivation.

The inoculated culture is incubated under favorable conditions for a time sufficient to build biomass. For microbial compositions for human use this is often at 37° C. temperature, pH, and other parameter with values similar to the normal human niche. The environment may be actively controlled, passively controlled (e.g., via buffers), or allowed to drift. For example, for anaerobic bacterial compositions, an anoxic/reducing environment may be employed. This can be accomplished by addition of reducing agents such as cysteine to the broth, and/or stripping it of oxygen. As an example, a culture of a bacterial composition may be grown at 37° C., pH 7, in the medium above, pre-reduced with 1 g/L cysteine-HCl.

When the culture has generated sufficient biomass, it may be preserved for banking. The organisms may be placed into a chemical milieu that protects from freezing (adding ‘cryoprotectants’), drying (‘lyoprotectants’), and/or osmotic shock (‘osmoprotectants’), dispensing into multiple (optionally identical) containers to create a uniform bank, and then treating the culture for preservation. Containers are generally impermeable and have closures that assure isolation from the environment. Cryopreservation treatment is accomplished by freezing a liquid at ultra-low temperatures (e.g., at or below −80° C.). Dried preservation removes water from the culture by evaporation (in the case of spray drying or ‘cool drying’) or by sublimation (e.g., for freeze drying, spray freeze drying). Removal of water improves long-term microbial composition storage stability at temperatures elevated above cryogenic conditions. Microbial composition banking may be done by culturing and preserving the strains individually, or by mixing the strains together to create a combined bank. As an example of cryopreservation, a microbial composition culture may be harvested by centrifugation to pellet the cells from the culture medium, the supernatant decanted and replaced with fresh culture broth containing 15% glycerol. The culture can then be aliquoted into 1 mL cryotubes, sealed, and placed at −80° C. for long-term viability retention. This procedure achieves acceptable viability upon recovery from frozen storage.

Microbial production may be conducted using similar culture steps to banking, including medium composition and culture conditions described above. It may be conducted at larger scales of operation, especially for clinical development or commercial production. At larger scales, there may be several subcultivations of the microbial composition prior to the final cultivation. At the end of cultivation, the culture is harvested to enable further formulation into a dosage form for administration. This can involve concentration, removal of undesirable medium components, and/or introduction into a chemical milieu that preserves the microbial composition and renders it acceptable for administration via the chosen route. For example, a microbial composition may be cultivated to a concentration of 10¹⁰ CFU/mL, then concentrated 20-fold by tangential flow microfiltration; the spent medium may be exchanged by diafiltering with a preservative medium consisting of 2% gelatin, 100 mM trehalose, and 10 mM sodium phosphate buffer. The suspension can then be freeze-dried to a powder and titrated.

After drying, the powder may be blended to an appropriate potency, and mixed with other cultures and/or a filler such as microcrystalline cellulose for consistency and ease of handling, and the bacterial composition formulated as provided herein.

In certain aspects, provided are bacterial compositions for administration in subjects. In some embodiments, the bacterial compositions are combined with additional active and/or inactive materials in order to produce a final product, which may be in single dosage unit or in a multi-dose format.

In some embodiments, the composition comprises at least one carbohydrate. A “carbohydrate” refers to a sugar or polymer of sugars. The terms “saccharide,” “polysaccharide,” “carbohydrate,” and “oligosaccharide” may be used interchangeably. Most carbohydrates are aldehydes or ketones with many hydroxyl groups, usually one on each carbon atom of the molecule. Carbohydrates generally have the molecular formula C_(n)H_(2n)O_(n). A carbohydrate may be a monosaccharide, a disaccharide, trisaccharide, oligosaccharide, or polysaccharide. The most basic carbohydrate is a monosaccharide, such as glucose, sucrose, galactose, mannose, ribose, arabinose, xylose, and fructose. Disaccharides are two joined monosaccharides. Exemplary disaccharides include sucrose, maltose, cellobiose, and lactose. Typically, an oligosaccharide includes between three and six monosaccharide units (e.g., raffinose, stachyose), and polysaccharides include six or more monosaccharide units. Exemplary polysaccharides include starch, glycogen, and cellulose. Carbohydrates may contain modified saccharide units such as 2′-deoxyribose wherein a hydroxyl group is removed, 2′-fluororibose wherein a hydroxyl group is replaced with a fluorine, or N-acetylglucosamine, a nitrogen-containing form of glucose (e.g., 2′-fluororibose, deoxyribose, and hexose). Carbohydrates may exist in many different forms, for example, conformers, cyclic forms, acyclic forms, stereoisomers, tautomers, anomers, and isomers.

In some embodiments, the composition comprises at least one lipid. As used herein, a “lipid” includes fats, oils, triglycerides, cholesterol, phospholipids, fatty acids in any form including free fatty acids. Fats, oils and fatty acids can be saturated, unsaturated (cis or trans) or partially unsaturated (cis or trans). In some embodiments the lipid comprises at least one fatty acid selected from lauric acid (12:0), myristic acid (14:0), palmitic acid (16:0), palmitoleic acid (16:1), margaric acid (17:0), heptadecenoic acid (17:1), stearic acid (18:0), oleic acid (18:1), linoleic acid (18:2), linolenic acid (18:3), octadecatetraenoic acid (18:4), arachidic acid (20:0), eicosenoic acid (20:1), eicosadienoic acid (20:2), eicosatetraenoic acid (20:4), eicosapentaenoic acid (20:5) (EPA), docosanoic acid (22:0), docosenoic acid (22:1), docosapentaenoic acid (22:5), docosahexaenoic acid (22:6) (DHA), and tetracosanoic acid (24:0). In some embodiments the composition comprises at least one modified lipid, for example a lipid that has been modified by cooking.

In some embodiments, the composition comprises at least one supplemental mineral or mineral source. Examples of minerals include, without limitation: chloride, sodium, calcium, iron, chromium, copper, iodine, zinc, magnesium, manganese, molybdenum, phosphorus, potassium, and selenium. Suitable forms of any of the foregoing minerals include soluble mineral salts, slightly soluble mineral salts, insoluble mineral salts, chelated minerals, mineral complexes, non-reactive minerals such as carbonyl minerals, and reduced minerals, and combinations thereof.

In some embodiments, the composition comprises at least one supplemental vitamin. The at least one vitamin can be fat-soluble or water soluble vitamins. Suitable vitamins include but are not limited to vitamin C, vitamin A, vitamin E, vitamin B12, vitamin K, riboflavin, niacin, vitamin D, vitamin B6, folic acid, pyridoxine, thiamine, pantothenic acid, and biotin. Suitable forms of any of the foregoing are salts of the vitamin, derivatives of the vitamin, compounds having the same or similar activity of the vitamin, and metabolites of the vitamin.

In some embodiments, the composition comprises an excipient. Non-limiting examples of suitable excipients include a buffering agent, a preservative, a stabilizer, a binder, a compaction agent, a lubricant, a dispersion enhancer, a disintegration agent, a flavoring agent, a sweetener, and a coloring agent.

In some embodiments, the excipient is a buffering agent. Non-limiting examples of suitable buffering agents include sodium citrate, magnesium carbonate, magnesium bicarbonate, calcium carbonate, and calcium bicarbonate.

In some embodiments, the excipient comprises a preservative. Non-limiting examples of suitable preservatives include antioxidants, such as alpha-tocopherol and ascorbate, and antimicrobials, such as parabens, chlorobutanol, and phenol.

In some embodiments, the composition comprises a binder as an excipient. Non-limiting examples of suitable binders include starches, pregelatinized starches, gelatin, polyvinylpyrolidone, cellulose, methylcellulose, sodium carboxymethylcellulose, ethylcellulose, polyacrylamides, polyvinyloxoazolidone, polyvinylalcohols, C12-C18 fatty acid alcohol, polyethylene glycol, polyols, saccharides, oligosaccharides, and combinations thereof.

In some embodiments, the composition comprises a lubricant as an excipient. Non-limiting examples of suitable lubricants include magnesium stearate, calcium stearate, zinc stearate, hydrogenated vegetable oils, sterotex, polyoxyethylene monostearate, talc, polyethyleneglycol, sodium benzoate, sodium lauryl sulfate, magnesium lauryl sulfate, and light mineral oil.

In some embodiments, the composition comprises a dispersion enhancer as an excipient. Non-limiting examples of suitable dispersants include starch, alginic acid, polyvinylpyrrolidones, guar gum, kaolin, bentonite, purified wood cellulose, sodium starch glycolate, isoamorphous silicate, and microcrystalline cellulose as high HLB emulsifier surfactants.

In some embodiments, the compositions of the present invention are combined with a carrier (e.g., a pharmaceutically acceptable carrier) which is physiologically compatible with the gastrointestinal tissue of the subject(s) to which it is administered. Carriers can be comprised of solid-based, dry materials for formulation into tablet, capsule or powdered form; or the carrier can be comprised of liquid or gel-based materials for formulations into liquid or gel forms. The specific type of carrier, as well as the final formulation depends, in part, upon the selected route(s) of administration. The therapeutic composition of the present invention may also include a variety of carriers and/or binders. In some embodiments, the carrier is micro-crystalline cellulose (MCC) added in an amount sufficient to complete the one gram dosage total weight. Carriers can be solid-based dry materials for formulations in tablet, capsule or powdered form, and can be liquid or gel-based materials for formulations in liquid or gel forms, which forms depend, in part, upon the routes of administration. Typical carriers for dry formulations include, but are not limited to: trehalose, malto-dextrin, rice flour, microcrystalline cellulose (MCC) magnesium sterate, inositol, FOS, GOS, dextrose, sucrose, and like carriers. Suitable liquid or gel-based carriers include but are not limited to: water and physiological salt solutions; urea; alcohols and derivatives (e.g., methanol, ethanol, propanol, butanol); glycols (e.g., ethylene glycol, propylene glycol, and the like). Preferably, water-based carriers possess a neutral pH value (i.e., pH 7.0). Other carriers or agents for administering the compositions described herein are known in the art, e.g., in U.S. Pat. No. 6,461,607.

In some embodiments, the composition comprises a disintegrant as an excipient. In some embodiments the disintegrant is a non-effervescent disintegrant. Non-limiting examples of suitable non-effervescent disintegrants include starches such as corn starch, potato starch, pregelatinized and modified starches thereof, sweeteners, clays, such as bentonite, micro-crystalline cellulose, alginates, sodium starch glycolate, gums such as agar, guar, locust bean, karaya, pectin, and tragacanth. In some embodiments the disintegrant is an effervescent disintegrant. Non-limiting examples of suitable effervescent disintegrants include sodium bicarbonate in combination with citric acid, and sodium bicarbonate in combination with tartaric acid.

In some embodiments, the bacterial formulation comprises an enteric coating or micro encapsulation. In certain embodiments, the enteric coating or micro encapsulation improves targeting to a desired region of the gastrointestinal tract. For example, in certain embodiments, the bacterial composition comprises an enteric coating and/or microcapsules that dissolves at a pH associated with a particular region of the gastrointestinal tract. In some embodiments, the enteric coating and/or microcapsules dissolve at a pH of about 5.5-6.2 to release in the duodenum, at a pH value of about 7.2-7.5 to release in the ileum, and/or at a pH value of about 5.6-6.2 to release in the colon. Exemplary enteric coatings and microcapsules are described, for example, in U.S. Pat. Pub. No. 2016/0022592, which is hereby incorporated by reference in its entirety.

In some embodiments, the composition is a food product (e.g., a food or beverage) such as a health food or beverage, a food or beverage for infants, a food or beverage for pregnant women, athletes, senior citizens or other specified group, a functional food, a beverage, a food or beverage for specified health use, a dietary supplement, a food or beverage for patients, or an animal feed. Specific examples of the foods and beverages include various beverages such as juices, refreshing beverages, tea beverages, drink preparations, jelly beverages, and functional beverages; alcoholic beverages such as beers; carbohydrate-containing foods such as rice food products, noodles, breads, and pastas; paste products such as fish hams, sausages, paste products of seafood; retort pouch products such as curries, food dressed with a thick starchy sauces, and Chinese soups; soups; dairy products such as milk, dairy beverages, ice creams, cheeses, and yogurts; fermented products such as fermented soybean pastes, yogurts, fermented beverages, and pickles; bean products; various confectionery products, including biscuits, cookies, and the like, candies, chewing gums, gummies, cold desserts including jellies, cream caramels, and frozen desserts; instant foods such as instant soups and instant soy-bean soups; microwavable foods; and the like. Further, the examples also include health foods and beverages prepared in the forms of powders, granules, tablets, capsules, liquids, pastes, and jellies. The composition may be a fermented food product, such as, but not limited to, a fermented milk product. Non-limiting examples of fermented food products include kombucha, sauerkraut, pickles, miso, tempeh, natto, kimchi, raw cheese, and yogurt. The composition may also be a food additive, such as, but not limited to, an acidulent (e.g., vinegar). Food additives can be divided into several groups based on their effects. Non-limiting examples of food additives include acidulents (e.g., vinegar, citric acid, tartaric acid, malic acid, fumaric acid, and lactic acid), acidity regulators, anticaking agents, antifoaming agents, foaming agents, antioxidants (e.g., vitamin C), bulking agents (e.g., starch), food coloring, fortifying agents, color retention agents, emulsifiers, flavors and flavor enhancers (e.g., monosodium glutamate), flour treatment agents, glazing agents, humectants, tracer gas, preservatives, stabilizers, sweeteners, and thickeners.

In certain embodiments, the bacteria disclosed herein are administered in conjunction with a prebiotic to the subject. Prebiotics are carbohydrates which are generally indigestible by a host animal and are selectively fermented or metabolized by bacteria. Prebiotics may be short-chain carbohydrates (e.g., oligosaccharides) and/or simple sugars (e.g., mono- and di-saccharides) and/or mucins (heavily glycosylated proteins) that alter the composition or metabolism of a microbiome in the host. The short chain carbohydrates are also referred to as oligosaccharides, and usually contain from 2 or 3 and up to 8, 9, 10, 15 or more sugar moieties. When prebiotics are introduced to a host, the prebiotics affect the bacteria within the host and do not directly affect the host. In certain aspects, a prebiotic composition can selectively stimulate the growth and/or activity of one of a limited number of bacteria in a host. Prebiotics include oligosaccharides such as fructooligosaccharides (FOS) (including inulin), galactooligosaccharides (GOS), trans-galactooligosaccharides, xylooligosaccharides (XOS), chitooligosaccharides (COS), soy oligosaccharides (e.g., stachyose and raffinose) gentiooligosaccharides, isomaltooligosaccharides, mannooligosaccharides, maltooligosaccharides and mannanoligosaccharides. Oligosaccharides are not necessarily single components, and can be mixtures containing oligosaccharides with different degrees of oligomerization, sometimes including the parent disaccharide and the monomeric sugars. Various types of oligosaccharides are found as natural components in many common foods, including fruits, vegetables, milk, and honey. Specific examples of oligosaccharides are lactulose, lactosucrose, palatinose, glycosyl sucrose, guar gum, gum Arabic, tagalose, amylose, amylopectin, pectin, xylan, and cyclodextrins. Prebiotics may also be purified or chemically or enzymatically synthesized.

Pharmaceutical Compositions

The compositions and methods of the present invention may be utilized to treat a subject in need thereof. In certain embodiments, the subject is a mammal such as a human, or a non-human mammal. When administered to subject, such as a human, the composition or the compound is preferably administered as a pharmaceutical composition comprising, for example, a compound of the invention and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as water or physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oils such as olive oil, or injectable organic esters. In preferred embodiments, when such pharmaceutical compositions are for human administration, particularly for invasive routes of administration (i.e., routes, such as injection or implantation, that circumvent transport or diffusion through an epithelial barrier), the aqueous solution is pyrogen-free, or substantially pyrogen-free. The excipients can be chosen, for example, to effect delayed release of an agent or to selectively target one or more cells, tissues or organs. The pharmaceutical composition can be in dosage unit form such as tablet, capsule (including sprinkle capsule and gelatin capsule), granule, lyophile for reconstitution, powder, solution, syrup, suppository, injection or the like. The composition can also be present in a transdermal delivery system, e.g., a skin patch. The composition can also be present in a solution suitable for topical administration, such as an eye drop.

A pharmaceutically acceptable carrier can contain physiologically acceptable agents that act, for example, to stabilize, increase solubility or to increase the absorption of a compound such as a compound of the invention. Such physiologically acceptable agents include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients. The choice of a pharmaceutically acceptable carrier, including a physiologically acceptable agent, depends, for example, on the route of administration of the composition. The preparation or pharmaceutical composition can be a self-emulsifying drug delivery system or a self-microemulsifying drug delivery system. The pharmaceutical composition (preparation) also can be a liposome or other polymer matrix, which can have incorporated therein, for example, a compound of the invention. Liposomes, for example, which comprise phospholipids or other lipids, are nontoxic, physiologically acceptable and metabolizable carriers that are relatively simple to make and administer.

The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of a subject without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

“Pharmaceutically acceptable salt” is used herein to refer to an acid addition salt or a basic addition salt which is suitable for or compatible with the treatment of patients.

The term “pharmaceutically acceptable acid addition salt” as used herein means any non-toxic organic or inorganic salt of the disclosed compounds. Illustrative inorganic acids which form suitable salts include hydrochloric, hydrobromic, sulfuric and phosphoric acids, as well as metal salts such as sodium monohydrogen orthophosphate and potassium hydrogen sulfate. Illustrative organic acids that form suitable salts include mono-, di-, and tricarboxylic acids such as glycolic, lactic, pyruvic, malonic, succinic, glutaric, fumaric, malic, tartaric, bitartaric, citric, ascorbic, maleic, benzoic, phenylacetic, cinnamic, salicylic, and sulfosalicylic acids, as well as sulfonic acids such as p-toluene sulfonic and methanesulfonic acids. Either the mono or di-acid salts can be formed, and such salts may exist in either a hydrated, solvated or substantially anhydrous form. In general, the acid addition salts of compounds disclosed herein are more soluble in water and various hydrophilic organic solvents, and generally demonstrate higher melting points in comparison to their free base forms. The selection of the appropriate salt will be known to one skilled in the art. Other non-pharmaceutically acceptable salts, e.g., oxalates, may be used, for example, in the isolation of compounds disclosed herein for laboratory use, or for subsequent conversion to a pharmaceutically acceptable acid addition salt.

The term “pharmaceutically acceptable basic addition salt” as used herein means any non-toxic organic or inorganic base addition salt of any acid compounds disclosed herein. Illustrative inorganic bases which form suitable salts include lithium, sodium, potassium, calcium, magnesium, or barium hydroxide. Illustrative organic bases which form suitable salts include aliphatic, alicyclic, or aromatic organic amines such as methylamine, trimethylamine and picoline or ammonia. The selection of the appropriate salt will be known to a person skilled in the art.

The phrase “pharmaceutically acceptable carrier” as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject. Some examples of materials which can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.

A pharmaceutical composition (preparation) can be administered to a subject by any of a number of routes of administration including, for example, orally (for example, drenches as in aqueous or non-aqueous solutions or suspensions, tablets, capsules (including sprinkle capsules and gelatin capsules), boluses, powders, granules, pastes for application to the tongue); absorption through the oral mucosa (e.g., sublingually); anally, rectally or vaginally (for example, as a pessary, cream or foam); parenterally (including intramuscularly, intravenously, subcutaneously or intrathecally as, for example, a sterile solution or suspension); nasally; intraperitoneally; subcutaneously; transdermally (for example as a patch applied to the skin); and topically (for example, as a cream, ointment or spray applied to the skin, or as an eye drop). The compound may also be formulated for inhalation. In certain embodiments, a compound may be simply dissolved or suspended in sterile water. Details of appropriate routes of administration and compositions suitable for same can be found in, for example, U.S. Pat. Nos. 6,110,973, 5,763,493, 5,731,000, 5,541,231, 5,427,798, 5,358,970 and 4,172,896, as well as in patents cited therein.

The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the subject being treated, the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 1 percent to about ninety-nine percent of active ingredient, preferably from about 5 percent to about 70 percent, most preferably from about 10 percent to about 30 percent.

Methods of preparing these formulations or compositions include the step of bringing into association an active compound, such as a compound of the invention, with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

Formulations of the invention suitable for oral administration may be in the form of capsules (including sprinkle capsules and gelatin capsules), cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), lyophile, powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a compound of the present invention as an active ingredient. Compositions or compounds may also be administered as a bolus, electuary or paste.

To prepare solid dosage forms for oral administration (capsules (including sprinkle capsules and gelatin capsules), tablets, pills, dragees, powders, granules and the like), the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; (10) complexing agents, such as, modified and unmodified cyclodextrins; and (11) coloring agents. In the case of capsules (including sprinkle capsules and gelatin capsules), tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.

A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceutical compositions, such as dragees, capsules (including sprinkle capsules and gelatin capsules), pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. The active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients.

Liquid dosage forms useful for oral administration include pharmaceutically acceptable emulsions, lyophiles for reconstitution, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, cyclodextrins and derivatives thereof, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

Formulations of the pharmaceutical compositions for rectal, vaginal, or urethral administration may be presented as a suppository, which may be prepared by mixing one or more active compounds with one or more suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound.

Formulations of the pharmaceutical compositions for administration to the mouth may be presented as a mouthwash, or an oral spray, or an oral ointment.

Alternatively or additionally, compositions can be formulated for delivery via a catheter, stent, wire, or other intraluminal device. Delivery via such devices may be especially useful for delivery to the bladder, urethra, ureter, rectum, or intestine.

Formulations which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate.

Dosage forms for the topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants that may be required.

The ointments, pastes, creams and gels may contain, in addition to an active compound, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to an active compound, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

Transdermal patches have the added advantage of providing controlled delivery of a compound of the present invention to the body. Such dosage forms can be made by dissolving or dispersing the active compound in the proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the compound in a polymer matrix or gel.

Ophthalmic formulations, eye ointments, powders, solutions and the like, are also contemplated as being within the scope of this invention. Exemplary ophthalmic formulations are described in U.S. Publication Nos. 2005/0080056, 2005/0059744, 2005/0031697 and 2005/004074 and U.S. Pat. No. 6,583,124, the contents of which are incorporated herein by reference. If desired, liquid ophthalmic formulations have properties similar to that of lacrimal fluids, aqueous humor or vitreous humor or are compatible with such fluids. A preferred route of administration is local administration (e.g., topical administration, such as eye drops, or administration via an implant).

The phrases “parenteral administration” and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, intraocular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.

Pharmaceutical compositions suitable for parenteral administration comprise one or more active compounds in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers that may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents that delay absorption such as aluminum monostearate and gelatin.

In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution, which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.

Injectable depot forms are made by forming microencapsulated matrices of the subject compounds in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissue.

For use in the methods of this invention, active compounds can be given per se or as a pharmaceutical composition containing, for example, about 0.1 to about 99.5% (more preferably, about 0.5 to about 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.

Methods of introduction may also be provided by rechargeable or biodegradable devices. Various slow release polymeric devices have been developed and tested in vivo in recent years for the controlled delivery of drugs, including proteinacious biopharmaceuticals. A variety of biocompatible polymers (including hydrogels), including both biodegradable and non-degradable polymers, can be used to form an implant for the sustained release of a compound at a particular target site.

Actual dosage levels of the active ingredients in the pharmaceutical compositions may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

The selected dosage level will depend upon a variety of factors including the activity of the particular compound or combination of compounds employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound(s) being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound(s) employed, the age, sex, weight, condition, general health and prior medical history of the subject being treated, and like factors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the therapeutically effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the pharmaceutical composition or compound at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. By “therapeutically effective amount” is meant the concentration of a compound that is sufficient to elicit the desired therapeutic effect. It is generally understood that the effective amount of the compound will vary according to the weight, sex, age, and medical history of the subject. Other factors which influence the effective amount may include, but are not limited to, the severity of the subject's condition, the disorder being treated, the stability of the compound, and, if desired, another type of therapeutic agent being administered with the compound of the invention. A larger total dose can be delivered by multiple administrations of the agent. Methods to determine efficacy and dosage are known to those skilled in the art (Isselbacher et al. (1996) Harrison's Principles of Internal Medicine 13 ed., 1814-1882, herein incorporated by reference).

In general, a suitable daily dose of an active compound used in the compositions and methods of the invention will be that amount of the compound that is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above.

If desired, the effective daily dose of the active compound may be administered as one, two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. In certain embodiments of the present invention, the active compound may be administered two or three times daily. In preferred embodiments, the active compound will be administered once daily.

In certain embodiments, compounds of the invention may be used alone or conjointly administered with another type of therapeutic agent.

In certain embodiments, conjoint administration of compounds of the invention with one or more additional therapeutic agent(s) provides improved efficacy relative to each individual administration of the compound of the invention or the one or more additional therapeutic agent(s). In certain such embodiments, the conjoint administration provides an additive effect, wherein an additive effect refers to the sum of each of the effects of individual administration of the compound of the invention and the one or more additional therapeutic agent(s).

This invention includes the use of pharmaceutically acceptable salts of compounds of the invention in the compositions and methods of the present invention. In certain embodiments, contemplated salts of the invention include, but are not limited to, alkyl, dialkyl, trialkyl or tetra-alkyl ammonium salts. In certain embodiments, contemplated salts of the invention include, but are not limited to, L-arginine, benenthamine, benzathine, betaine, calcium hydroxide, choline, deanol, diethanolamine, diethylamine, 2-(diethylamino)ethanol, ethanolamine, ethylenediamine, N-methylglucamine, hydrabamine, 1H-imidazole, lithium, L-lysine, magnesium, 4-(2-hydroxyethyl)morpholine, piperazine, potassium, 1-(2-hydroxyethyl)pyrrolidine, sodium, triethanolamine, tromethamine, and zinc salts. In certain embodiments, contemplated salts of the invention include, but are not limited to, Na, Ca, K, Mg, Zn or other metal salts.

The pharmaceutically acceptable acid addition salts can also exist as various solvates, such as with water, methanol, ethanol, dimethylformamide, and the like. Mixtures of such solvates can also be prepared. The source of such solvate can be from the solvent of crystallization, inherent in the solvent of preparation or crystallization, or adventitious to such solvent.

Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal-chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

EXAMPLES Example 1: The Maternal Microbiome Modulates Fetal Neurodevelopment and Offspring Sensory Behaviour Summary

“Dysbiosis” of the maternal gut microbiome, in response to environmental challenges such as infection, altered diet and stress during pregnancy, has been increasingly associated with abnormalities in offspring brain function and behavior. However, whether the maternal gut microbiome regulates neurodevelopment in the absence of environmental challenge remains unclear. In addition, whether the maternal microbiome exerts such influences during critical periods of embryonic brain development is poorly understood. Here we investigate how depletion, and selective colonization, of the maternal gut microbiota influences fetal neurodevelopment. Embryos from antibiotic-treated and germ-free dams exhibit widespread transcriptomic alterations in the fetal brain relative to conventionally-colonized controls, with reduced expression of several genes involved in axonogenesis. In addition, embryos from microbiome-depleted mothers exhibit deficient thalamocortical axons and impaired thalamic axon outgrowth in response to cell-extrinsic guidance cues and growth factors. Consistent with the importance of fetal thalamocortical axonogenesis for shaping sensory processing neural circuits, restricted depletion of the maternal microbiome from pre-conception through mid-gestation yields offspring that exhibit tactile hyposensitivity in sensorimotor behavioral tasks. Gnotobiotic colonization of antibiotic-treated dams with a limited consortium of bacteria indigenous to the gut microbiome prevents abnormalities in fetal brain gene expression, fetal thalamocortical axonogenesis and adult tactile sensory behavior associated with maternal microbiome depletion. Metabolomic profiling reveals that the maternal microbiome regulates levels of numerous small molecules in the maternal serum as well as the brains of fetal offspring. Select microbiome-dependent metabolites—trimethylamine N-oxide, 5-aminovalerate, imidazole propionate, and hippurate—sufficiently promote axon outgrowth from fetal thalamic explants. Moreover, maternal supplementation with the metabolites during early gestation abrogates deficiencies in fetal thalamocortical axons and prevents abnormalities in tactile sensory behavior in offspring from microbiome-depleted dams. Altogether, these findings reveal that the maternal gut microbiome promotes fetal thalamocortical axonogenesis and offspring sensory behavior, likely by direct signaling of microbially modulated metabolites to neurons in the developing brain.

Results and Discussion

The intestinal microbiome is an important modulator of brain function and behavior¹. However, whether the maternal gut microbiome impacts the brain development during prenatal critical periods is poorly understood. Various model organisms reared devoid of microbial colonization (germ-free, GF) or depleted of the gut microbiome (antibiotic-treated, ABX) exhibit altered neurophysiology and behavior compared to conventionally-colonized (specific pathogen-free, SPF) controls²⁻⁴. Interestingly, only a subset of phenotypes can be corrected by postnatal restoration of the microbiome⁵⁻⁸, suggesting a role for the early life microbiome in regulating developmental processes that impact brain function and behavior in adulthood. Indeed, in animal models, the gut microbiome is required for mediating adverse effects of maternal challenges, such as immune activation^(9,10), high fat diet⁶ and psychosocial stress^(11,12), on neurobehavioral abnormalities in adult offspring. It remains unclear, however, whether such microbial influences on neurodevelopment originate antenatally, via disrupted function of the maternal microbiome, and/or postnatally, via vertically transmitted alterations in the neonatal microbiome¹³⁻¹⁵. Moreover, while existing studies report that the maternal gut microbiome can modulate host responses to acute dietary-, stress- or inflammation-related insults, whether it impacts offspring development in the absence of environmental challenges requires investigation. Herein, we examine roles for the maternal gut microbiome during homeostasis in regulating early embryonic brain development and later-life behavior of the offspring.

To determine whether the maternal microbiome influences fetal neurodevelopment, we first examined fetal brains from offspring of murine dams that were reared SPF, GF, or treated with broad-spectrum ABX to deplete the maternal gut microbiome from pre-conception through midgestation [embryonic day (E)14.5]. Transcriptomic profiling revealed that depletion of the maternal microbiome altered the expression of 333 genes in fetal brains of E14.5 embryos, including many involved in axonogenesis (FIG. 1A, Table 1). Gene ontology analysis indicated that these genes were relevant for cell proliferation, cell junction, cell-matrix adhesion and cellular developmental processes (FIG. 2A). The 160 downregulated genes mapped to protein interaction networks that included those relevant to axon guidance, Wnt signaling, cell morphogenesis, neuronal differentiation and glutamatergic synapse (FIG. 2C), whereas the 173 upregulated genes mapped to networks that included those relevant to apoptosis, long-term depression, cell adhesion and GABAergic synapse (FIG. 2D). Validation by qPCR revealed consistent downregulation of Netrin-G1 a (NTNG1), a glycosylphosphatidylinositol-tethered protein highly expressed by developing thalamocortical axons¹⁶, in fetal brains from offspring of both ABX and GF dams (FIG. 2B). Consistent with observed reductions in NTNG1 transcript (FIG. 1A, FIG. 2C), fetal brain sections from E14.5 offspring of both ABX and GF dams exhibited reduced Netrin-G1a+immunoreactivity localized to thalamocortical neurons (FIGS. 1B-1C, FIGS. 3A-3I). In addition, evaluation of three-dimensional representations of Netrin-G1a+thalamocortical axons in cleared whole embryonic brains revealed decreased axonal volume and length in E14.5 offspring from microbiome-depleted dams, with corresponding increases in distances from the leading axon to the cortex and reduced circumference of the axonal bundle at the internal capsule (FIGS. 1E-1I). Notably, fetal brains from ABX offspring displayed decreased L1+ axonal immunoreactivity compared to SPF control, but no significant differences in neuronal DAPI levels in the thalamus (FIG. 1D, FIGS. 31 and 4A-4E). This suggests that the reductions in Netrin-G1a expression reflect decreases in thalamocortical axonal projections, rather than diminished receptor expression on existing axons or the absence of thalamic neurons themselves.

These findings align with recent studies reporting reductions in adult axonal markers in the cortex and myenteric plexus in response to microbiota depletion¹⁷⁻²⁰ (Example 3). Overall, results from these experiments suggest that the maternal microbiome is required to support fetal thalamocortical axonogenesis in the developing offspring.

Axonogenesis involves cell intrinsic and extrinsic factors that work in concert to direct axon polarity, elongation and pathfinding. To gain insight into whether the reductions in Netrin-G1a+thalamocortical axons seen in response to maternal microbiome depletion were due to impaired axon formation, deficient axon guidance, or both, we cultured E14.5 thalamic explants, either alone or in the presence of endogenous cues from striatal and hypothalamic explants^(21,22). Monoculture of E14.5 thalamic explants from offspring of either SPF or ABX dams resulted in substantial axon outgrowth (FIGS. 5A-5C), suggesting that the reductions in Netrin-Ole axons seen in embryos of microbiome-depleted dams are not due to an intrinsic inability of the thalamus to form or elongate axons. Indeed, thalamic neurons from embryos of ABX dams generated increased numbers of axons when grown in cell culture matrices containing growth factors, with no significant difference in axon length, as compared to SPF controls (FIGS. 5A-5C); this suggests enhanced capacity for axon formation, but not elongation, in fetal thalamic neurons from ABX dams that are grown in rich media. However, in response to co-culture with fetal striatal and hypothalamic explants from ABX dams, fetal thalamic neurons from embryos of ABX dams exhibited impaired axon outgrowth, with decreased number and length of axons as compared to co-cultured control explants from SPF mothers (FIGS. 1J-1M, white vs. black). These abnormalities in cue-mediated axonal outgrowth were observed for thalamic axons proximal to the striatal explant (FIGS. 1J-1M, white vs. black), which produces growth promoting and attractive guidance cues^(23,24), as well as axons proximal to the hypothalamic explant (FIGS. 5D-5F, white vs. black), which produces growth-inhibiting and repulsive guidance cues^(25,26). Taken together, these results indicate that fetal thalamic neurons from E14.5 offspring of ABX dams display deficient axon outgrowth in response to cell-extrinsic tissue-derived factors.

To gain further insight into whether depletion of the maternal microbiome alters tissue-derived cues to impair axon outgrowth, fetal thalamic explants from E14.5 embryos of SPF or ABX dams were co-cultured with striatal and hypothalamic explants from the contrasting experimental group. When thalamic explants from E14.5 embryos of SPF dams were co-cultured with fetal striatal and hypothalamic explants from offspring of ABX dams, there were no significant differences in the number or length of axons from SPF thalamic neurons proximal to the ABX striatal (FIGS. 1J-1M; purple in the original image vs. black) or hypothalamic explants (FIGS. 5D-5F; purple in the original image vs. black). This suggests that tissue-derived factors from ABX dams sufficiently support axon outgrowth from SPF thalamic neurons. In contrast, when thalamic explants from E14.5 embryos of ABX dams were co-cultured with fetal striatal and hypothalamic explants from offspring of SPF dams, fetal thalamic neurons from ABX offspring exhibited deficiencies in axon outgrowth, at levels similar to those seen in response to co-culture with ABX tissues (FIGS. 1J-1M, FIGS. 5D-5F; teal in the original image vs. white). This suggests that endogenous soluble factors from SPF explants are not sufficient to correct impairments in axon outgrowth of ABX thalamic neurons, and that ABX thalamic neurons display incorrect responses to factors from SPF tissues. Such impairments in axon outgrowth in response to tissue-derived cues could be attributed to erroneously repulsive responses to attractive guidance cues^(27,28), hyperresponsiveness to repulsive cues²⁹ and/or cue-induced disruptions in responses to neurotrophic factors present in the culture media³⁰. Overall, these findings indicate that tissue-derived cues are necessary but not sufficient for mediating maternal microbiota-dependent reductions in thalamic axonogenesis and further suggest that depletion of the maternal microbiome impairs responses of embryonic thalamocortical neurons to axonogenic cues.

From prenatal through early postnatal development, thalamocortical axons are guided to the somatosensory cortex, where they form dense synaptic contacts with layer 4 neurons to mediate sensory processing³¹⁻³⁴. To gain insight into whether microbiome-induced alterations in fetal thalamocortical axonogenesis confer lasting influences on offspring behavior, SPF dams were treated with ABX or vehicle from pre-conception through E14.5, and then colonized with a conventional SPF microbiome for the remainder of gestation through offspring postnatal development (FIG. 6A). Conventionalized offspring of ABX- or vehicle-treated dams were tested in a battery of sensory behavioral tasks (FIGS. 6A-6G, FIGS. 7A-7F and 8A-8F). In the von Frey filament test for hindpaw sensorimotor function^(35,36), adult offspring of ABX dams required significantly increased force thresholds for paw withdrawal in response to hindpaw stimulation compared to control offspring from SPF dams (FIGS. 6B-6C), suggesting impaired tactile sensation. Consistent with this, in the adhesive removal test for forepaw sensorimotor function³⁷, adult offspring of ABX dams exhibited significantly increased latency to detect and contact the forepaw stimulus compared to control offspring from SPF dams (FIGS. 6D-6E). There was no difference in the time taken to remove the adhesive after first contact (FIG. 6F), suggesting that ABX offspring exhibit deficient initial paw tactile sensation, but no disruption in motor response (FIG. 6G). Statistically significant effects of the maternal microbiome on offspring tactile sensory behavior were observed when data were averaged by litter to represent individual dams as biological replicates (FIGS. 6A-6G), as well as when data from individual offspring were analyzed (FIGS. 7A-7C). There was no statistically significant difference in behavioral performance between male and female mice in these tasks (FIGS. 7D-7F). In addition, abnormalities in sensory behavior appeared to be limited to paw tactile responses, as there were no differences in behavioral performance between ABX and control SPF offspring in the hot plate test for thermosensory behavior³⁸, the visual cliff test for visual sensory behavior³⁹, the whisker-dependent texture discrimination test for vibrissae sensory perception⁴⁰, the rotarod test for motor coordination⁴¹ and the prepulse inhibition task for acoustic startle response and sensorimotor gating⁴² (FIG. 8A-8F). Altogether, these results demonstrate that depletion of the maternal gut microbiome during early to mid-gestation impairs fetal thalamocortical axonogenesis and yields adult offspring with disrupted neurobehavioral responses to forepaw and hindpaw tactile stimuli.

The gut microbiome is comprised of several hundred different bacterial taxa, many of which exhibit specialized functions and differential interactions with host physiology”⁴³⁻⁴⁶. To determine whether the effects of the maternal microbiome on offspring neurodevelopment and behavior are mediated by particular bacterial taxa, we colonized ABX-treated dams during preconception with a consortium of bacteria representing one of the two dominant phyla of the gut microbiota—Firmicutes and Bacteroidetes (FIGS. 9A-10B and 11A, Tables 2 and 3). Colonization of ABX-treated dams with Clostridia-dominant spore-forming bacteria (Sp) of the phylum Firmicutes abrogated many adverse effects of maternal microbiota depletion on fetal brain gene expression and thalamocortical axon outgrowth (FIGS. 10A-10M). E14.5 fetal brains from embryos of Sp-colonized dams exhibited transcriptomic profiles that clustered closely with samples derived from SPF controls, with restored expression of many genes relevant to axon guidance (FIG. 10A; FIGS. 9C-9F, Table 1). Notably, reductions in NTNG1 expression and Netrin-G1a+thalamocortical axons observed in response to maternal microbiome depletion were prevented by maternal colonization with Sp bacteria (FIGS. 10B-10J, FIGS. 3A-3I and 4A-4E, FIGS. 9C-9F). In contrast, colonizing ABX-treated dams with a consortium of Bacteroides (BD), containing B. thetaiotaomicron, B. uniformis, B. vulgatus, B. ovatus and B. fragilis (FIG. 11A), conferred only a modest increase in Netrin-G1a+thalamocortical axons in fetal brains from E14.5 offspring, which exhibited statistical significance by group, but not across individual rostral to caudal sections compared to ABX controls (FIGS. 11B-11D). Fetal thalamic explants from E14.5 embryos of Sp-colonized dams also exhibited significantly increased axon outgrowth compared to controls from ABX-treated dams (FIGS. 5G-5L). Deficiencies in paw tactile sensory behavior in the adhesive removal and von Frey filament tests seen in adult offspring of ABX-treated dams were also prevented by maternal colonization with Sp bacteria (FIGS. 10K-10M), with no differences in performance between males and females and in other sensory behavioral tasks (FIGS. 7A-7F and 8A-8F). Overall, these findings suggest that limited bacterial taxa, including Sp bacteria in particular, are sufficient to prevent the adverse effects of maternal microbiota depletion on fetal thalamocortical axonogenesis and offspring sensory behavior.

The gut microbiome modulates the bioavailability of hundreds of biochemicals in the circulating blood^(8,47-49). During pregnancy, the maternal intrauterine environment supplies nutrients and growth factors to nurture offspring growth, which is particularly important for the rapidly developing fetal brain^(50,51). The blood brain barrier begins forming at E16.5 and continues developing during the first three weeks of postnatal life^(52,53), rendering the developing fetal brain permeable to circulating metabolites. Based on our finding that the maternal microbiota is important for regulating fetal neurodevelopment, we hypothesized that the maternal microbiome regulates maternal circulating metabolites and thereby conditions metabolite profiles in the fetus. To investigate this, we performed tandem liquid chromatography mass spectrometry to globally profile biochemicals in maternal sera and fetal brain lysates from SPF, ABX, GF and Sp-colonized dams on E14.5 of gestation. A total of 753 metabolites were identified in maternal sera and 567 in fetal brain lysates, spanning amino acid, carbohydrate, co-factor and vitamin, energy, lipid, nucleotide, peptide and xenobiotic biochemical super pathways (Tables 4 and 5). Supervised hierarchical clustering of samples based on differential levels of maternal blood metabolites led to co-clustering of samples derived from SPF and Sp dams compared to GF and ABX dams (FIG. 12A). Metabolomic profiles in maternal blood from ABX and GF mice clustered closely by principal component analysis (PCA), whereas those from SPF and Sp-colonized dams formed a separate co-cluster (FIG. 12B). This suggests that Sp bacteria recapitulate many of the effects of the SPF microbiota on maternal blood biochemical profiles, and further aligns with the phenotypic similarities between offspring of ABX and GF dams versus SPF and Sp dams in fetal axonogenesis and adult sensory behavior. Random Forests analysis identified 30 maternal blood metabolites that discriminate maternal microbiota status with 100% predictive accuracy (FIG. 13A). Overall, these data reveal widespread effects of the maternal microbiome on circulating serum biochemicals during pregnancy.

Interestingly, metabolomic profiles from fetal brain lysates of SPF dams clustered away from profiles from fetal brain lysates of Sp-colonized, ABX, and GF dams (FIG. 12C), suggesting that there are global alterations in fetal brain metabolomic profiles from E14.5 fetal brains of offspring from gnotobiotic mothers. 165 fetal brain metabolites were commonly downregulated in embryos from ABX and GF dams, relative to SPF controls (FIG. 12D, Table 4). 27 fetal brain metabolites were commonly downregulated in embryos from ABX and GF dams, relative to Sp controls (FIG. 12E, Table 4). Pathway analysis revealed alterations in several amino acid, lipid, and xenobiotic metabolites in fetal brain lysates from ABX and GF dams compared to SPF and Sp dams (FIGS. 12F-12G, FIG. 13C). Random Forests analysis identified the top 30 fetal brain metabolites that were predictive with 87.5% accuracy of maternal SPF and Sp versus ABX and GF microbiota status (FIG. 12H). 22 metabolites were similarly and significantly decreased in fetal brain lysates from ABX and GF dams relative to both SPF and Sp dams (Table 5). Of these 22 fetal brain metabolites, 8 were similarly differentially regulated in maternal sera from ABX and GF dams compared to SPF and Sp controls (Table 5), suggesting that the maternal microbiome modulates the bioavailability of these metabolites in maternal blood with direct effects on the bioavailability of the same metabolites in fetal brain. Overall, these findings reveal that the maternal microbiome modulates biochemical profiles and select metabolites in the fetal brains of developing offspring.

To further determine whether particular microbiota-dependent metabolites in the fetal brain mediate the ability of the maternal microbiome to promote fetal thalamocortical axonogenesis, thalamic explants from E14.5 embryos of ABX-treated dams were exposed to physiologically-relevant levels of select fetal brain biochemicals, and axon outgrowth was evaluated ex vivo. The metabolites trimethylamine-N-oxide (TMAO), N, N, N-trimethyl-5-aminovalerate (TMAV), imidazole propionate (IP), 3-indoxyl sulfate (3-IS) and hippurate (HIP) were selected based on their>2-fold reduction in both maternal blood and fetal brain lysates from ABX and GF dams, relative to SPF controls, and their restoration to SPF levels by maternal colonization with Sp bacteria (FIG. 12I, FIG. 13B). In addition, each metabolite is known to be regulated in adult stool, blood and/or prefrontal cortex by the gut microbiome^(8,47,54,55). Fetal thalamic explants harvested from E14.5 embryos of ABX dams exhibited impaired axonogenesis in response to co-culture with ABX striatal and hypothalamic explants, as previously described (FIGS. 1J-1M, FIG. 5A-5L, white vs. black). Notably, exposure to physiologically-relevant concentrations of TMAO, 5-AV, IP or HIP, but not 3-IS, significantly increased axon number to levels seen in fetal brain explants from embryos of SPF dams (FIGS. 14A-14C, FIG. 15A). 5-AV and IP also significantly increased axon length, whereas TMAO and 3-IS induced modest, but not statistically significant, increases in axon length, while HIP had no effect (FIG. 15B). No statistically significant changes were found in number and length of ABX thalamic axons proximal to hypothalamus in response to TMAO, 5-AV, IP, 3-IS, or HIP (FIGS. 15C-15D). To further test whether maternal metabolite supplementation impacts fetal neurodevelopment in vivo, ABX-treated dams were injected intraperitoneally with a cocktail of TMAO, 5-AV, IP, and HIP metabolites (4-MM) or vehicle from E7-14 of gestation, the developmental time frame during which thalamocortical axonogenesis occurs^(56,57) Metabolite dosages were calculated based on maternal serum metabolomic data and physiological concentrations reported in literature to reflect daily levels needed to achieve those observed in SPF dams (see Methods section). Notably, maternal supplementation with 4-MM prevented the reductions in Netrin-G1 thalamocortical axons seen with maternal microbiome depletion (FIGS. 14D-14F, FIG. 16A-16D). Consistent with results from the axon outgrowth assay, these findings suggest that select microbial metabolites, including 4-MM, are important for promoting fetal thalamocortical axonogenesis. Furthermore, adult offspring of ABX dams that were supplemented with 4 MM exhibited improvements in tactile sensory behavior in the von Frey filament and adhesive removal tasks, relative to vehicle-treated ABX controls, which were statistically significant when analyzed by dam (FIGS. 14G-141) as well as by individual offspring (FIGS. 17A-17C). There were no significant differences observed between male and female mice in these behavioral tasks (FIGS. 17D-17F). Altogether, results from this study reveal that the maternal microbiome promotes fetal thalamocortical axonogenesis and postnatal tactile sensory behavior, likely via microbiome-dependent biochemicals, such as TMAO, 5-AV, IP, and HIP, in the fetal brain.

The gut microbiome modulates numerous bioactive molecules in the intestine, serum and various extraintestinal organs^(54,58,59). Findings from this work reveal that during pregnancy, the maternal gut microbiome regulates metabolites, not only in the maternal compartment, but also in the fetus itself, including the embryonic brain. Select fetal brain metabolites that are regulated by the maternal gut microbiome induce axon outgrowth from thalamic explants and promote fetal thalamocortical axonogenesis and adult tactile sensory behavior in offspring of microbiome-depleted dams. While the molecular mechanisms underlying the effects of select microbial metabolites on neurons remain unclear, some metabolites, such as TMAO, TMAV and HIP, have been associated with neurological conditions and factors related to neurite outgrowth⁶⁰⁻⁶⁵ (Example 3). In addition, findings from this study parallel recent evidence that malnutrition-induced alterations in the maternal microbiome were associated with reduced white matter in the brains of adolescent and adult offspring and that inflammation-induced alterations in the maternal gut microbiome disrupted somatosensory cortical architecture in adult offspring^(20,66-69.) Furthermore, a recent study of microbiomes in malnourished children reported that children with severe acute malnutrition exhibited dysregulation of several proteins associated with axonogenesis, including semaphorins, neurotrophins, netrin, slit and ephrin, which were ameliorated by treatment with microbiota-directed diets”⁷⁰. Results presented herein support an important role for the maternal microbiome in promoting offspring neurodevelopment, and further suggest that interactions between the microbiome and nervous system begin prenatally through influences of the maternal gut microbiome on fetal brain metabolomic profiles and gene expression. Altogether, findings from this study identify early to mid-gestation as a critical period during which the maternal microbiome promotes fetal neurodevelopment to support developmental processes underlying adult tactile sensory behavior.

Example 2: Methods Used for Example 1 Mice

C57Bl/6J mice were purchased from Jackson Laboratories, reared as SPF or rederived as GF, and bred in flexible film isolators at the UCLA Center for Health Sciences barrier facility. Animals were maintained on a 12-h light-dark schedule in a temperature-controlled environment with autoclaved “breeder” chow (Lab Diets 5K52) and standard chow (Lab Diet 5010) and autoclaved water provided ad libitum.

Sample Size Determination

6-8 week-old mice were randomly assigned to experimental groups, which included age- and sex-matched cohorts of males and females for timed matings. Given that maternal microbiome status is the primary experimental variable across experiments, biological sample sizes reflect independent dams. Experiments evaluating fetal outcomes include at least 2 randomly selected embryos per dam, where data from offspring from a single dam were averaged to represent the dam as the biological “n”. For behavioral assays, all offspring were behaviorally tested and data from offspring from the same dam were averaged to represent the dam as the biological “n”. These data are presented in FIGS. 1A-1M, 6A-6G, 10A-10M, 12A-121, and 14A-141, whereas behavioral data per individual offspring are presented in the other figures. All experiments were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals using protocols approved by the Institutional Animal Care and Use Committee at UCLA.

Antibiotic Treatment and Conventionalization

4-5 week old SPF mice were gavaged twice daily (08:00 and 17:00) for 1 week with a cocktail of neomycin (100 mg/kg), metronidazole (100 mg/kg), and vancomycin (50 mg/kg), according to methods previously described to mimic GF status⁷¹. Ampicillin (1 mg/ml) was provided ad libitum in drinking water. Breeders were then paired and time-mated, where up to 2 additional weeks were required to conception. Gestational day 0.5 was determined by observation of copulation plug. Dams were then separated and maintained on ABX drinking water until E14.5 to preclude the daily stress of oral gavage in pregnant dams (1 mg/ml ampicillin, 1 mg/ml neomycin, and 0.5 mg/ml vancomycin; metronidazole was excluded due to its confounding bitter taste). Fecal samples from ABX-treated dams were collected and plated anaerobically on Schaedler's broth and tryptic soy agar to confirm bacterial clearance. For behavioral assays, pregnant dams were conventionalized at E14.5 with SPF bedding that was gathered from a male and female C57Bl/6J cage⁷². Pregnant dams were maintained in SPF bedding for the remainder of gestation, and offspring were reared with SPF bedding, added weekly, until behavioral testing. Conventionalization was validated by fecal 16S rDNA sequencing, as described in the “16S rDNA sequencing” section below.

Gnotobiotic Colonization

Mice were treated with ABX as described in the “antibiotic treatment” section above, then given sterile water and orally gavaged 1 day later with Sp or BD bacteria. Sp-colonized mice were generated as previously described′. Briefly, fecal pellets from C57Bl/6J SPF mice were freshly suspended in a 10× volume of pre-reduced PBS in an anaerobic chamber. Chloroform was added to 3% (vol/vol), the sample was shaken vigorously and incubated at 37° C. for 1 hr. Chloroform was removed by percolation with CO₂ from a compressed cylinder. 200 ul of the resultant suspension was orally gavaged into adult GF C57Bl/6J “founder” mice housed in designated gnotobiotic isolators. Fecal samples were collected from the Sp mice at >2 weeks after gavage and suspended at 50 mg/ml in pre-reduced PBS. 200 ul of the suspension was orally gavaged to ABX-treated experimental mice. For the Bacteroides (BD) consortium, B. thetaiotaomicron (ATCC 29148), B. vulgatus (ATCC 8482) and B. uniformis (ATCC 8492), B. ovatus (ATCC 8483) and B. fragilis (NCTC 9343) were grown in Brain Heart Infusion media (BD Biosciences) supplemented with 5 μg/ml hemin (Frontier Scientific) and 0.5 μg/ml vitamin K1 (Sigma Aldrich) under anaerobic conditions. A 200 μl suspension of 1:1:1:1:1 OD of each strain was orally gavaged into ABX-treated mice. Colonization status was validated by 16S rDNA sequencing of fecal samples collected on E14.5 (FIGS. 9B and 11A). For BD, total relative abundance of Bacteroides was 95.24%, and individual species distributions were determined by qPCR as B. thetaiotaomicron: 9.38%, B. vulgatus: 18.75% and B. uniformis: 15.63%, B. ovatus: 46.88% and B. fragilis: 9.38%.

Fetal Brain RNA Sequencing

Dams were sacrificed on E14.5 by cervical dislocation to preclude confounding effects of CO₂ on maternal and fetal physiology. Embryonic brains were microdissected from SPF, ABX, and Sp colonized mice and placed in Trizol (Invitrogen). RNA was extracted using the RNAeasy Mini kit with on-column genomic DNA-digest (Qiagen), and cDNA synthesis was performed using the qScript cDNA synthesis kit (Quantabio). RNA quality of RIN>8.0 was confirmed using the 4200 Tapestation system (Agilent). RNA was prepared using the TruSeq RNA Library Prep kit and 2×69 bp paired-end sequencing was performed using the Illumina HiSeq 4000 platform by the UCLA Neuroscience Genomics Core. FastQC v0.11.8 and HiSAT2 2.1.0^(74,75) were used for quality filtering and mapping. Reads were aligned to UCSC Genome Browser assembly ID: mm10. Differential expression analysis was conducted using DESeq2 1.24.0⁷⁶. Heatmaps were generated using the pheatmap package for R. GO term enrichment analysis of differentially expressed genes with q<0.05 was conducted using DAVID v6.8⁷⁷.

Quantitative RT-PCR

Dams were sacrificed on E14.5 by cervical dislocation to preclude confounding effects of CO₂ on maternal and fetal physiology. Embryonic brains were microdissected on E14.5 and sonicated in Trizol for RNA isolation using the RNAeasy Mini kit with on-column genomic DNA-digest (Qiagen). cDNA synthesis was performed using the qScript cDNA synthesis kit (Quantabio). qRT-PCR was performed on a QuantStudio 5 thermocycler (ThermoFisher Scientific) using SYBR green master mix with Rox passive reference dye and validated primer sets obtained from Primerbank (Harvard).

Axon Outgrowth Assay

Dams were sacrificed on E14.5 by cervical dislocation to preclude confounding effects of CO₂ on maternal and fetal physiology. Thalamic, striatal, and hypothalamic explants were isolated from E14.5 embryonic brains and transferred to ice-cold HBSS (Invitrogen). Explants were sliced to ˜500 μm and placed on a thin layer of 50 μl BD Matrigel (Beckton Dickinson) on a 15 mm coverslip. Each coverslip contained a thalamic explant at the center and a striatal and hypothalamic explant on each side, at 1 mm equidistant from the thalamic explant. Explants were incubated in warmed neurobasal complete media containing 1× neurobasal medium (Thermofisher Scientific), 1× GlutaMax (Thermofisher Scientific), and 2% B-27 (Thermofisher Scientific) for 48 hrs at 37° C., and fed with fresh media every 24 hrs. After 48 hrs, media was gently aspirated and replaced with 4% PFA for 1 hr and processed for immunofluorescence staining with 1:500 (3 tubulin III anti-mouse antibody (EMD Millipore MAB1637). Axons were imaged using a Leica DMi8 epifluorescence microscope and quantified using Fiji software⁷⁸. Axon numbers were quantified per 200 μm of thalamus at a distance of 200 μm from the thalamus. Length of axons was quantified by averaging length of the 10 longest axons proximal to striatum or hypothalamus. Data for number and length of axons in the explant co-culture system was normalized by subtraction of data from monoculture of thalamic explants from the corresponding experimental group.

Metabolite Supplementation

Thalamic, striatal, and hypothalamic explants from ABX-treated dams were harvested and cultured as described in the “axon outgrowth assay” section above. For metabolite treatment, BD Matrigel was supplemented with 10 1.1M, 100 nM, or 1 nM of trimethylamine-N-oxide (TMAO), 5-aminovalerate (5-AV), imidazole propionate (IP), 3-indoxyl-sulfate (3-IS), or hippurate (llip)^(48,79-81). 5-aminovalerate is a precursor to N,N,N-trimethyl-5-aminovalerate (TMAV), which is not commercially available, and both are implicated in carnitine metabolism^(82,83). Metabolite concentrations were determined as physiologically relevant, based on reported concentrations detected in blood and/or cerebrum from the mouse multiple tissue metabolomic database (MMIvIDB), human metabolome database (HMDB) and existing literature^(48,79-81). Axons were stained, imaged and analyzed as described in the “axon outgrowth assay” section above.

In Vivo Metabolite Supplementation

To test the effects of TMAO, 5-AV, IP, and HIP on fetal axonogenesis and behavior, physiological concentrations of metabolites were administered intraperitoneally as a single dose per each day in order to limit stress to pregnant dams. Controls were injected with vehicle. Metabolite concentrations were calculated based on physiological levels reported in mouse blood^(48,79-81), total blood volume of pregnant mouse dams (approximately 58.5 ml/kg⁸⁴), and relative reductions observed in maternal sera of ABX dams compared SPF dams (Table 5). The metabolite mixture (4-MM): 121 ug (TMAO), 9 ug (5-AV), 92 ug (IP), and 2 ug (HIP) in 200 ul of 0.1 M PBS was injected intraperitoneally into E7.5 ABX dams once a day for 7 days. To assess fetal thalamocortical axon levels, dams treated with 4-MM or vehicle were sacrificed on E14.5, and fetal brains were harvested and processed as described in the “immunofluorescence staining” and “modified CLARITY” sections below. To test for behavioral effects, E14.5 pregnant dams were taken off antibiotic water (ANV) on E14.5, transferred into cages with sterile water, and conventionalized as described in the “conventionalization” section above. Adult offspring (P42-P56) were tested in the von Frey filament test and adhesive removal test as described in the “behavioral assay” section below.

Immunofluorescence Staining

E14.5 embryos were fixed in 4% paraformaldehyde for 24 hrs at 4° C., cryoprotected in 30% sucrose 24 hrs at 4° C. and sectioned at 10 μm using a Leica CM1950 cryostat. Sections were blocked with 10% donkey serum for 1 hr. Primary antibodies were diluted in 3% donkey serum and incubated for 15-18 hrs at 4° C. with Netrin-G1a anti-goat antibody (1:100, R&D Systems, AF1166) or Neural Cell Adhesion Molecule L1 anti-rat antibody (1:500, EMD Millipore, MAB5272). Sections were then incubated for 2 hrs at room temperature in their corresponding donkey anti-goat and anti-rat secondary antibodies conjugated to Alexa Fluor 568 or 488 (1:1000, Thermofisher Scientific). Images were acquired using the Zeiss Axio Examiner LSM 780 confocal microscope. Rostral to caudal sections were adjusted in Fiji: process>noise>despeckle, to remove non-specific staining. Fluorescence intensity of stains was quantified in Fiji based on a set region of interest drawn to encompass Netrin-G1a staining observed in SPF samples.

Tissue Clearing and Imaging

E14.5 embryos were collected and fixed in 4% paraformaldehyde for 48 hours at 4° C. Tissue was rendered transparent using methods for CLARITY-based clearing⁸⁵ with the following modifications. Tissues were incubated in a hydrogel solution containing 4% paraformaldehyde, 4% acrylamide (Bio-Rad), 0.05% bis-acrylamide (Bio-Rad), 0.25% VA-044 (A4P4B0.05) for 3 days at 4° C. Prior to hydrogel polymerization, the solution was exchanged with new solution lacking bis-acrylamide and paraformaldehyde (A4PO4) and polymerized at 37° C. for 3 hrs. Samples were passively cleared in 8% SDS for 2 weeks at 42° C., and then incubated with primary antibodies (Netrin-G1 a anti-goat (1:100, R&D Systems, AF1166) and L1 anti-rat (1:500, EMD Millipore, MAB5272)) for 1 week at 25° C. Samples were washed and then incubated in secondary antibodies (1:1000, Thermofisher Scientific) for 5 days at 25° C. Samples were equilibrated for 15-18 hrs in a histodenz-based refractive index matching solution (RI 1.47; Sigma Aldrich, D2158) and imaged on a Zeiss LSM 780 with 488 or 561 nm illumination using a 5× objective with 3 um z-slices. Images were adjusted for brightness and contrast post hoc using Arivis Vision4D v3.0. 3D reconstructions were optically z-sliced for quantification of stain volume, length of axons, circumference of internal capsule and distance of rostral axon tip to cortical surface. Positively stained areas of interest were segmented and visualized using CTAn and CTVol software packages (Bruker Corporation), respectively.

Behavioral Assays

For behavioral assays, investigators were blinded to experimental groups. For each behavioral test, cages were brought to the testing room at least 30 minutes before testing to enable acclimation and reduce stress. Equipment and testing chambers were thoroughly cleaned with Accel disinfectant (Unimed) before and after each trial.

Adhesive Removal Test

The adhesive removal test was performed according to methods adapted from Bouet et al. 2009³⁷. Briefly, mice were acclimated to the testing cage for 5 min A small adhesive tape (0.3 cm×0.4 cm) was gently applied to both forepaws, and mice were returned to the testing cage. Mice were observed for contact time, as defined as the latency to which the mouse reacts to the presence of the adhesive tape, and for removal time, as defined as the latency to which the mouse removes both pieces of tape completely. Contact time and removal time were manually recorded using a standard lab multi-timer by experimenters blinded to the mouse experimental group.

Von Frey Filament Test

The von Frey filament test was performed according to methods adapted from Dixon et al., 1980⁸⁶. Briefly, mice were placed on a wide gauge, wire mesh surface in a testing chamber and acclimated for 10 minutes daily for two consecutive days prior to testing day. On the testing day, mice were placed in the testing chamber, acclimated for 10 minutes, and von Frey filaments were applied from the underside of the mesh to the plantar surface of the hindpaw. The process is repeated with increasing gauges (0.4, 0.6, 1, 1.4, 2, 4, 6 grams of force) of von Frey filaments until stimulation elicits a hindpaw withdrawal, wherein the mouse responds by flicking its paw away from the stimulus. Upon paw withdrawal, the next weaker stimulus is defined as threshold. Responses of up-down paw stimulation were manually recorded and analyzed according to the Chaplan Method of 50% paw withdrawal threshold³⁶.

Prepulse Inhibition Test

The prepulse inhibition test was performed to measure sensorimotor gating⁸⁷. Mice were placed in a restraint tube mounted on a startle measuring platform (San Diego Instruments) and acclimated to the testing chamber for 10 minutes. White noise is presented in the recording chamber for 5 minutes, followed by 6 startle presentations and a pseudorandomized prepulse inhibition phase, which consisted of either no startle, 120 db startle stimulus only, or 70 db prepulse with startle, 75 db prepulse with startle, or 80 db prepulse with startle. Acoustic startle was recorded with a pliezo-electric sensory, and the percent prepulse inhibition was defined as: [((the startle stimulus only−prepulse with startle)/startle stimulus only)*100].

Hot Plate Test

To test for somatic pain response¹, mice were acclimated to a clear plastic cylinder for 30 s, then placed on an advanced hot plate (VWR) that was heated to 52° C. The latency to show nociceptive response as indicated by a paw lick, paw flick, vocalization, or a jump was recorded, and mouse was immediately returned to the home cage.

Novel Whisker Texture Test

The whisker texture test was performed according to methods adapted from Wu et al., 2013². Mice were habituated in 50 cm×50 cm white plexiglass testing chamber for 10 minutes for 2 consecutive days. On testing day, mice were first subjected to a learning phase in which they were placed in the testing chamber for 5 minutes with two objects of identical texture (aluminum oxide sand paper, 80 grit). Mice were then returned to home cage for 5 minutes. In the test phase, mice were placed back into chamber with two objects, one with the original texture (80 grit) and one with new texture (220 grit). The trials were recorded with an overhead video camera and Ethovision software (Noldus) was used to analyze number of times and duration spent investigating the novel and familiar textures.

Visual Cliff Avoidance Test

To assess depth perception and visual impairment³, mice were placed in a 42.5 cm×60 cm clear plexiglass testing chamber on top of a 3 ft×4 ft rectangular table. One third of the chamber hung over the edge of the table to create a visual effect of a cliff drop-off at a height of 3 ft. Mice were placed in the middle of the chamber 10 times. Mice were given 5 minutes to either exit off the platform towards the table or toward the cliff side of the chamber. Each choice was recorded and averaged by an experimenter blinded to mouse experimental group.

Rotarod Test

To test for motor coordination and balance⁴, mice were placed in one of 4 compartments in a rotarod apparatus (Rotamex, Columbus Instruments) consisting of a cylinder that rotates speeds accelerating from 5 rpm to 60 rpm in 300 seconds. On the first day, mice acclimated to the apparatus with no rotation for 2 minutes. On the testing day, mice were returned to the apparatus and rotation was initiated. Latency to fall and final speed achieved by the accelerating rod before falling was detected by an infrared sensor and recorded. Mice were tested three times and scores were averaged.

16S rDNA Sequencing

Bacterial genomic DNA was extracted from mouse fecal samples using the MoBio PowerSoil Kit. The library was generated according to methods adapted from Caporaso et al. 2011⁸⁸. The V4 regions of the 16S rRNA gene were PCR amplified using individually barcoded universal primers and 30 ng of the extracted genomic DNA. The PCR reaction was set up in triplicate, and the PCR product was purified using the Qiaquick PCR purification kit (Qiagen). 250 ng of purified PCR product from each sample were pooled and sequenced by Laragen, Inc. using the Illumina MiSeq platform and 2×250 bp reagent kit for paired-end sequencing. Operational taxonomic units (OTUs) were chosen by open reference OTU picking based on 97% sequence similarity to the Greengenes 13_5 database. Taxonomy assignment and rarefaction were performed using QIIME1.8.0⁸⁹.

Metabolomics

At E14.5 maternal serum was collected by cardiac puncture, separated using SST vacutainer tubes (Beckton Dickinson) and frozen at −80° C. Embryonic brains were collected and immediately snap frozen in liquid nitrogen. Each fetal brain sample consisted of 5 embryonic brains pooled from the same litter. Samples were prepared using the automated MicroLab STAR system (Hamilton Company) and analyzed on GC/MS, LC/MS and LC/MS/MS platforms by Metabolon, Inc. Protein fractions were removed by serial extractions with organic aqueous solvents, concentrated using a TurboVap system (Zymark) and vacuum dried. For LC/MS and LC-MS/MS, samples were reconstituted in acidic or basic LC-compatible solvents containing >11 injection standards and run on a Waters ACQUITY UPLC and Thermo-Finnigan LTQ mass spectrometer, with a linear ion-trap frontend and a Fourier transform ion cyclotron resonance mass spectrometer back-end. For GC/MS, samples were derivatized under dried nitrogen using bistrimethyl-silyl-trifluoroacetamide and analyzed on a Thermo-Finnigan Trace DSQ fast-scanning single-quadrupole mass spectrometer using electron impact ionization. Chemical entities were identified by comparison to metabolomic library entries of purified standards. Following log transformation and imputation with minimum observed values for each compound, data were analyzed using one-way ANOVA to test for group effects. P and q-values were calculated based on two-way ANOVA contrasts. Principal components analysis was used to visualize variance distributions. Supervised Random Forest analysis was conducted to identify metabolomics prediction accuracies. Volcano plots were generated using R, with differentially regulated metabolites at q<0.05.

Statistical Methods

Statistical analysis was performed using Prism software (GraphPad). Data were assessed for normal distribution and plotted in the figures as mean±SEM. For each figure, n=the number of independent maternal biological replicates. For assessments involving fetal brains, each maternal biological sample reflects an average of 2-5 embryo “technical” replicates. For behavioral assessments, all offspring were tested. Data for littermates from the same dam were averaged and presented in the FIGS. 1A-1M, 6A-6G, 10A-10M, 12A-12I, and 14A-14I with n=independent maternal dams; individual data for each offspring are provided in the other figures. No samples or animals were excluded from the analyses. Differences among >2 groups with only one variable were assessed using one-way ANOVA with Tukey's post hoc test. Taxonomic comparisons from 16S rDNA sequencing analysis were analyzed by Kruskal-Wallis test with Tukey's post hoc test. Two-way ANOVA with Tukey's post-hoc test was used for >2 groups with two variables. Significant differences emerging from the above tests are indicated in the figures by *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Notable non-significant differences are indicated in the figures by “n.s.”.

Example 3: Additional Discussion Regarding Example 1

Gut Microbiota Modulation of Axons and Myelination

Results from this study reveal that the maternal microbiome promotes fetal thalamocortical axonogenesis. We observe deficiencies in L1 and Netrin-G1a expression in E14.5 brains of ABX dams (FIGS. 1B-1D). Consistent with a previous report, we observe only modest changes in L1 expression in E14.5 brains of fetuses from GF dams relative to SPF controls⁹⁰ (FIGS. 1B-1D), pointing to potential compensatory effects of GF rearing as compared to acute microbiome depletion. Our finding extends several previous reports that the gut microbiota modulates axon structure and axonal myelination in adult animals. Adolescent (4 weeks) and adult (12 weeks) GF mice display reduced white matter structure in the corpus callosum, anterior commissure and internal capsule²⁰, which is a defining anatomical structure for thalamocortical axon projections (FIGS. 1E-1I). Consistent with this, brains of GF mice also exhibit reduced expression of neuronal (NeuN), axonal (neurofilament-L), and myelination (MBP) markers relative to control mice colonized with a healthy human microbiota¹⁹. In a recent ultrastructural study, microbiota depleted mice exhibited reduced axon diameter and increased myelination in the brain′. Finally, children aged 6-36 months with healthy microbiome exhibit increased expression of central nervous system development proteins compared to children with severe acute malnutrition microbiomes. These proteins are associated with axonogenesis, including semaphorins (SEMA3A, SEMA5A, SEMA6A, SEMA6B), neurotrophins (NTRK2, NTRK3), netrin (UNC5D), slit (SLITRK5) and ephrin (EFNA5), which were ameliorated by treatment with microbiota-directed diets”. Microbiome-dependent alterations in axons may extend beyond the brain itself, as adult GF and ABX-treated mice exhibited reduced axonal innervations of the colonic epithelium¹⁷ and ABX treatment of a mouse model of multiple sclerosis increased axon numbers in the spinal cord⁹². Altogether, we propose that the maternal gut microbiome during pregnancy plays an important role in regulating fetal thalamocortical axonogenesis,

Potential Mechanisms of Microbially Modulated Metabolites

Results from this study indicate that the maternal microbiome modulates numerous biochemicals in the fetal brain, and that select metabolites—TMAO, 5-AV, IP, and HIP—promote fetal thalamocortical axonogenesis and offspring tactile sensory behavior. While microbiome-dependent regulation of TMAO, 5-AV, IP and HIP has been reported across metabolomic datasets for adult mouse and human blood, urine, and/or intestine^(48,54,93,94), little is known regarding the functional roles for each metabolite on host physiology.

Dysregulation of TMAO, produced through a two-step enzymatic process performed by gut microbes and the liver, has been implicated in metabolic, cardiovascular, cerebrovascular, stroke and Alzheimer's disease^(62,64,95) Although a cognate receptor for TMAO has not been described, TMAO is reported to modulate glucocorticoid receptors and the Gi3y subunit of GPCRs, to promote protein stability and folding as an organic osmolyte, and to regulate the phosphorylation of insulin-like growth factor 2 (IGF2)⁹⁶⁻⁹⁹. Such effects on IGF2 have been reported to increase sympathetic neurite outgrowth¹⁰⁰, which could be relevant to the observed axonogenic effects of TMAO on thalamocortical neurons.

TMAV is metabolized from dietary glycine and is associated with glucose metabolism and diets rich in whole grain^(82,94,101). Increases in TMAV have been associated with type 1 diabetes with microalbuminuria and metformin-treated type 2 diabetes^(102,103), but the relation of TMAV to axon or brain development has not been previously reported. 5-AV, a precursor for TMAV, is microbially produced from L-lysine. L-lysine monooxygenase (DavB) and 5-aminovaleramide amidohydrolase (DavA) are key enzymes in the 5-AV pathway, whereby DavB catalyzes the oxidation of L-lysine to produce 5-aminovaleramide; DavA then converts 5-aminovaleramide into 5-AV^(104,105). 5-AV has been shown to negatively regulate baclofen, a GABAB receptor agonist, to suppress naloxone-stimulated luteinizing hormone-releasing hormone¹⁰⁶. Further, 5-AV has been associated with reductions in the inhibitory effect of baclofen on norepinephrine release from noradrenergic terminals¹⁰⁶. Separately, application of 5-AV onto rat hippocampal slices reduced pyramidal cell GABAB-mediated ICE inhibitory postsynaptic potential (IPSP)¹⁰⁷. Though the exact mechanism by which 5-AV alters axon outgrowth is unclear, one hypothesis is that the influences of 5-AV on GABAB receptors, which are key regulators of synaptic release and axonal trafficking^(108,109), can impact cortical neuronal migration and axon/dendrite morphological maturation by modulating cAMP signaling^(110,111).

IP is a product of direct microbial, but not murine, metabolism⁴⁷. IP is a microbial metabolite derived from histidine and has been reported to impair insulin signaling through mTORC1⁴⁸. IP is associated with nonalcoholic fatty liver disease and is a potential inducer of steatosis and hepatic inflammation^(112,113). In another study, IP was found in urine of IBS patients 112,113. There have been no previous reports of IP regulation of axon development, however, activation of mTOR has been shown to increase axonal growth capacity¹¹⁴ and promote axon regeneration after injury or disease^(115,116).

HIP, synthesized through glycine conjugation with benzoate in the liver, is a metabolite of folic acid, which affects neural tube formation and brain development⁶⁵. Decreased excretion of HIP has been described in patients with schizophrenia, depression, stroke, autism, and gastrointestinal disorder, and in animal models of acute and chronic stress¹¹⁷⁻¹¹⁹. While HIP has not been previously implicated in axon development, the HIP precursor, benzoate, has been shown to have anti-inflammatory properties and to reduce microglial and astroglial inflammatory responses in the experimental autoimmune encephalomyelitits (EAE) model of multiple sclerosis¹²⁰.

Overall, a few previous reports have associated altered microbiome-related metabolites with altered neurodevelopment and adverse neurological outcomes. However, extensive further research is needed to uncover functional roles for microbially-modulated metabolites on host health and disease, and to identify the molecular and cellular mechanisms underlying the axonogenic effects of TMAO, 5-AV, IP and HIP on thalamic neurons.

Example 4: References for Examples 1-3

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Example 5: Table 1 for Examples 1-3, Provided as Parts Tables 1A and 1B

Tables 1A and 1B (collectively “Table 1”) relate to genes differentially regulated in fetal brains from E14.5 offspring of SPF, ABX or Sp dams [log 2(fold change), p<0.05].

TABLE 1A gene SPF1 SPF2 SPF3 ABX1 ABX2 ABχ3 1700017B05Rik −0.013691 −0.002052 0.015593 0.025156 0.058023 0.028209 1810055G02Rik −0.061343 0.014947 0.044343 0.197809 0.145103 0.091112 2610015P09Rik −0.011342 0.011362 −0.000109 −0.021900 −0.004519 −0.017002 2610020C07Rik −0.014458 −0.001735 0.016030 0.050565 0.046883 0.061797 2700046A07Rik −0.009163 −0.004508 0.013571 0.023998 0.044703 0.049588 2810408I11Rik 0.007824 −0.005805 −0.002053 −0.010604 −0.013178 −0.013934 2810468N07Rik −0.004334 0.002402 0.001923 −0.008982 0.002462 0.016393 3830406C13Rik −0.001768 0.000824 0.000942 0.044060 0.044487 0.034581 4930447A16Rik −0.013183 0.001102 0.011971 −0.018683 −0.030010 −0.023630 4930511A08Rik 0.000961 −0.001008 0.000046 0.023827 0.024982 0.028427 4933439C10Rik 0.005944 −0.005483 −0.000484 −0.035563 −0.021005 −0.034008 5930403L14Rik −0.007801 −0.012516 0.020101 0.009663 0.040193 0.038374 6330415G19Rik 0.023579 −0.023759 −0.000208 −0.029023 −0.023986 −0.032611 6330549D23Rik −0.002816 0.008622 −0.005846 −0.000361 −0.003263 0.008310 6430573F11Rik −0.002783 0.003907 −0.001133 0.030136 0.035753 0.047103 6530402F18Rik −0.009169 −0.019152 0.027893 0.016504 0.006367 0.035088 9130019P16Rik 0.008037 −0.003473 −0.004598 0.041812 −0.004955 0.026020 9430065F17Rik 0.006487 −0.004295 −0.002215 0.040504 0.034990 0.043795 A330069E16Rik 0.021457 0.000467 −0.022255 −0.039767 −0.021666 −0.028666 A930003O13Rik 0.008534 −0.004075 −0.004498 0.031827 0.020610 0.015188 Acad9 0.014002 −0.004695 −0.009413 0.011991 −0.017004 0.017679 Acsbg1 −0.001349 0.011006 −0.009733 0.035657 0.019684 0.025965 Acsf2 −0.014025 0.009749 0.004169 0.045994 0.027336 0.014967 Adamts8 −0.005227 −0.004093 0.009275 0.038792 0.005886 0.003352 Agap3 −0.004007 0.002240 0.001759 0.006894 0.015122 0.019847 Airn −0.014368 0.024494 −0.010444 0.060721 0.016651 −0.007511 Ak7 0.006079 −0.015814 0.009604 0.026037 0.027584 0.033285 Aldoc −0.008723 0.003713 0.004970 0.050519 0.042087 0.025349 Alpl −0.026255 0.032967 −0.007348 0.063191 −0.003241 0.009526 Ankrd11 −0.007986 −0.002796 0.010717 0.009465 0.021113 0.019980 Ankrd6 0.010002 −0.008794 −0.001270 0.009612 0.021290 0.033858 Ap1s1 −0.005761 0.004385 0.001357 0.011485 −0.023600 −0.026905 Ap3s1 0.026425 −0.021761 −0.005080 −0.016582 −0.033174 −0.032931 Arhgap10 −0.010300 −0.001443 0.011658 0.036992 0.026512 0.048041 Arid1a −0.006357 −0.013669 0.019811 0.013765 0.017112 0.037745 Arl6ip1 −0.005105 0.005298 −0.000212 0.019671 0.028489 0.009925 Armt1 −0.005414 0.000823 0.004573 −0.018029 −0.007078 −0.012271 Arrb2 0.000805 0.000221 −0.001027 0.010841 0.029075 0.035742 Arsa −0.013241 0.014601 −0.001495 0.020089 0.002528 0.001705 Arxes2 0.034379 −0.017030 −0.017974 −0.019434 −0.002059 0.000409 Ate1 0.008769 −0.008560 −0.000260 −0.023434 −0.021123 −0.017943 Atg4d −0.016432 0.023325 −0.007194 0.022272 0.004867 0.009955 Atn1 −0.013192 0.027587 −0.014796 0.035722 0.022566 −0.011805 Atp5h 0.001910 −0.002401 0.000488 −0.020770 −0.010187 −0.019857 Atp6ap2 0.011443 −0.007599 −0.003915 −0.028920 −0.020698 −0.019311 Atp6v1b2 0.000791 −0.002553 0.001759 −0.018028 −0.020296 −0.018980 Atp8b1 −0.000387 0.000071 0.000316 0.034696 0.036054 0.005977 Atpaf1 −0.000147 0.005012 −0.004881 0.035659 0.031654 0.018562 Atxn2 0.011466 −0.021734 0.010024 0.024880 0.027287 0.055831 Aven −0.006599 0.003708 0.002868 0.028375 0.039584 0.014862 AW209491 0.000205 0.007619 −0.007866 0.037580 0.045194 0.032102 AW549877 0.026798 −0.010848 −0.016333 −0.027213 −0.008327 −0.012785 Axin2 0.005063 −0.011303 0.006174 0.019507 0.039106 0.049395 B230307C23Rik 0.023502 −0.013659 −0.010136 0.018593 0.011025 −0.002458 B3galt2 0.025716 −0.015715 −0.010354 −0.025490 −0.024071 −0.028417 B4galt4 0.000734 0.000744 −0.001479 −0.018939 −0.004451 −0.045745 Bahcc1 −0.009021 0.017805 −0.008950 0.033934 0.040962 0.048132 Baiap2l1 −0.006255 −0.005406 0.011591 0.026503 0.020592 0.032249 BC020402 0.009038 −0.011837 0.002720 −0.027330 −0.042233 −0.023153 BC024139 −0.016769 0.039915 −0.023999 0.037852 0.027411 0.046107 Blzf1 0.005958 −0.019611 0.013445 0.028175 0.040626 0.041971 Bola1 −0.018362 0.007986 0.010201 0.003714 0.008970 0.049348 Brd4 0.003335 −0.021785 0.018168 0.037761 0.012085 0.025806 Brox 0.012174 0.003414 −0.015729 −0.026016 −0.010931 −0.034093 C1galt1c1 −0.000854 0.001332 −0.000478 0.016391 −0.015485 −0.002103 C1ql3 −0.004347 −0.002109 0.006433 −0.017748 0.007530 0.002770 C330006A16Rik −0.015080 0.023234 −0.008445 0.044709 0.012585 0.016892 C77080 −0.004162 0.003333 0.000819 0.013053 −0.006343 0.031779 C77370 0.027254 −0.013995 −0.013650 −0.025613 −0.017690 −0.037493 Cadm2 0.040193 0.002283 −0.043698 −0.030855 −0.021279 −0.064849 Camk1d 0.000337 −0.012317 0.011879 −0.015921 −0.043106 −0.038512 Camk2d 0.004607 0.030487 −0.035868 −0.010691 −0.031988 −0.053633 Capn3 0.027683 0.018873 −0.047729 0.014403 0.043743 0.075704 Capns2 0.004877 −0.004142 −0.000750 0.038492 −0.000046 0.026689 Card9 −0.003258 0.007833 −0.004607 −0.003916 0.014272 0.011576 Carns1 −0.004446 0.017186 −0.012907 0.006516 0.026279 0.010374 Cartpt −0.002567 0.007039 −0.004499 0.004503 −0.008416 0.003896 Casp9 −0.014370 0.008107 0.006156 0.015928 −0.005713 0.015670 Casz1 −0.022234 0.021494 0.000409 0.060426 0.024529 0.027321 Cc2d1a −0.257968 0.083484 0.142951 0.335001 0.272811 0.208523 Ccbl2 0.003477 0.003221 −0.006721 −0.026898 −0.037175 −0.014036 Ccdc102a −0.031371 0.017031 0.013834 0.035437 0.051464 0.014786 Ccdc137 0.008489 −0.005540 −0.002988 −0.038536 −0.019412 −0.029305 Ccdc162 0.002568 −0.006305 0.003717 0.007030 0.018654 0.026689 Ccdc18 −0.000572 −0.008631 0.009148 −0.007838 0.006226 −0.032179 Ccdc71l 0.005088 0.000476 −0.005584 0.022939 0.031096 0.004786 Ccdc88b −0.011836 0.001540 0.010211 0.017762 0.017957 −0.001311 Ccne1 0.002665 −0.012001 0.009254 0.039234 0.020589 0.039957 Cd34 −0.003419 0.006727 −0.003332 −0.036170 −0.016832 −0.025070 Cdc14b 0.009504 −0.006412 −0.003141 0.006548 0.013176 0.040384 Cdca2 −0.266534 0.070071 0.162103 0.411964 0.319834 0.252383 Cdh20 0.008465 0.002678 −0.011214 0.020026 0.032882 0.042300 Cdh24 −0.017059 −0.005905 0.022672 0.031148 0.016972 0.044253 Cdk6 −0.003797 0.001883 0.001906 0.007143 0.007508 0.019817 Cdr1 −0.016600 0.003017 0.013422 0.058517 0.040913 0.022276 Celsr2 −0.025725 0.030497 −0.005334 0.043664 0.062178 0.014182 Cenpb −0.017236 0.007523 0.009559 0.014506 0.021959 0.032934 Cep250 −0.013055 −0.005236 0.018109 0.019258 0.021353 0.019959 Cep85l −0.003983 0.012911 −0.009019 0.015615 0.028534 0.053004 Cerk −0.010242 0.009021 0.001156 0.002380 0.010758 0.014197 Chml 0.009298 −0.001418 −0.007932 0.031669 0.035364 0.044170 Cit −0.018667 −0.016421 0.034460 0.021447 0.019356 0.055051 Cklf −0.008200 −0.009877 0.017908 −0.014710 0.010322 −0.015076 Cks1b −0.001601 0.016342 −0.014911 0.001054 0.005292 0.003786 Clic3 −0.005059 −0.002885 0.007911 0.011067 0.008871 0.015261 Clic6 0.002859 0.021987 −0.025237 −0.047145 −0.018982 −0.052512 Clmn −0.009099 −0.005537 0.014523 0.082723 −0.002874 0.014205 Clock 0.014457 −0.002232 −0.012353 −0.014854 −0.008199 −0.024915 Cmc1 −0.018338 −0.000061 0.018168 0.004406 0.049142 0.051443 Cntnap5b −0.016586 0.008286 0.008158 0.003131 0.008968 0.008780 Coch −0.001445 0.018071 −0.016839 0.026409 0.030949 0.038882 Col7a1 −0.002193 −0.002355 0.004537 0.017110 0.018107 0.023445 Commd9 −0.002758 0.007875 −0.005150 −0.021648 −0.023572 −0.003816 Cplx2 0.017446 −0.017357 −0.000299 0.021516 0.008766 0.016710 Cpped1 0.032194 −0.005421 −0.027405 −0.057051 −0.027413 −0.015832 Cramp1l −0.004812 −0.012143 0.016798 0.019807 0.017316 0.035009 Creb3l1 0.000881 −0.009094 0.008160 0.022693 0.024629 0.027213 Creb5 0.006540 −0.017713 0.011008 0.000149 0.012897 0.039606 Crebrf 0.009161 −0.011623 0.002384 −0.023723 −0.019569 −0.023265 Cryab −0.002842 0.002140 0.000697 0.021712 0.031529 0.028885 Cryzl1 −0.009324 −0.008925 0.018077 0.023165 0.022803 0.057894 Ctrl −0.017782 0.017056 0.000515 −0.012540 −0.013972 −0.007952 Ctsa −0.021223 0.017967 0.002985 0.029892 0.040181 0.008847 Ctss 0.004135 0.008322 −0.012541 0.023067 0.037548 0.010570 Cuedc1 0.004721 0.008828 −0.013648 0.023554 0.031999 0.025011 Cyb561 −0.025178 0.009752 0.015095 −0.008772 −0.042851 −0.036849 Cyb5a 0.008189 0.004498 −0.012774 0.039605 0.030557 0.008247 Cycs −0.008731 0.004820 0.003872 0.019464 0.029763 0.038929 Cyp27b1 −0.009895 0.016250 −0.006495 0.005391 0.032703 0.031313 D030068K23Rik 0.014014 −0.006894 −0.007222 0.014070 0.008443 0.025314 D11Wsu47e 0.016323 −0.014643 −0.001848 0.026474 0.015762 0.009628 Dbx2 −0.009244 0.022217 −0.013236 −0.000435 0.030839 0.016024 Ddx25 −0.020833 0.014566 0.006031 0.044811 0.051798 0.024080 Ddx27 −0.002956 0.005537 −0.002596 0.030404 0.034883 0.032498 Deb1 −0.001152 −0.011867 0.012911 −0.019768 −0.032047 −0.030629 Dennd1c 0.000212 −0.002196 0.001981 0.006279 −0.004790 0.005813 Dennd2a −0.014606 0.001585 0.012888 0.018377 0.020808 0.029888 Dimt1 0.013622 −0.006121 −0.007598 −0.020443 −0.019826 −0.010126 Dio3os −0.012568 −0.002213 0.014650 −0.033654 −0.031932 −0.025854 Dlx5 −0.014187 0.002352 0.011716 0.024353 0.034172 0.027894 Dmd 0.001628 0.005958 −0.007619 0.031223 0.015562 0.012080 Dnaaf2 −0.001262 −0.013143 0.014274 0.023126 0.038542 0.027179 Dnah8 −0.008404 0.023940 −0.015847 −0.016825 −0.037441 −0.047572 Dnase1 −0.008909 −0.006020 0.014812 0.041121 0.026113 0.026609 Dok2 −0.013260 0.004552 0.008614 0.037157 0.033690 0.013780 Dpp10 0.019643 −0.002160 −0.017727 −0.029691 −0.032823 −0.036993 Dpy19l3 0.009264 −0.018870 0.009422 −0.049102 −0.026817 −0.012825 E130012A19Rik −0.000807 0.000399 0.000407 0.005042 0.029057 0.041743 E130307A14Rik 0.007925 −0.013096 0.005081 0.009247 0.006031 0.007514 E2f8 0.004428 −0.003275 −0.001165 0.005423 0.044469 0.036104 Ece1 −0.009918 −0.009007 0.018741 0.049753 0.025776 0.016607 Efhb −0.024910 0.013484 0.011106 0.000963 0.007163 0.031476 Elk4 0.002294 0.009720 −0.012100 0.032681 0.027819 0.031420 Emp2 0.017869 −0.025995 0.007762 0.051533 0.021452 0.042750 En2 −0.000753 −0.006176 0.006898 0.019659 0.023398 0.034555 Epas1 −0.024938 0.022076 0.002475 0.021593 0.030437 0.036091 Ephb2 −0.015079 −0.004089 0.018958 0.009054 0.022220 0.034428 Epor −0.019207 −0.003296 0.022200 0.036909 0.025510 0.048726 Erbb2 −0.018271 0.015545 0.002525 0.044054 0.015948 0.011309 Esd −0.004985 0.002110 0.002862 −0.025372 −0.028868 −0.009583 Exo5 0.007018 −0.010454 0.003377 −0.034123 −0.001907 −0.009558 Exph5 0.004080 0.006158 −0.010294 0.062642 0.020350 0.009826 F11r −0.016389 0.009750 0.006498 0.046693 0.031773 0.016056 F8 0.007068 0.005149 −0.012296 −0.011191 −0.040813 −0.050165 Fah 0.009051 −0.004302 −0.004792 −0.012398 −0.032889 −0.038826 Fam122b −0.008536 0.009272 −0.000791 0.014354 −0.016323 −0.015643 Fam126a 0.008034 −0.006720 −0.001353 −0.008075 −0.012619 0.000457 Fam160b2 −0.008582 0.008630 −0.000100 0.022511 −0.003434 0.006279 Fam199x 0.023852 0.003553 −0.027875 −0.004559 −0.010494 −0.045552 Fam19a1 −0.030689 0.011926 0.018274 0.041559 0.028945 0.054458 Fam207a −0.006397 0.005546 0.000825 −0.021166 −0.014093 0.000665 Fam208a −0.020957 −0.007026 0.027550 0.040359 0.027260 0.034140 Fam20a 0.002328 0.002379 −0.004718 0.005715 0.021642 0.020349 Fam43b −0.010221 −0.010278 0.020284 0.027201 0.025763 0.044832 Fam76b −0.000292 0.011086 −0.010878 0.019934 −0.016597 0.014410 Fgf1 −0.010076 0.026548 −0.016851 −0.030351 −0.016212 −0.028811 Fkbp1b −0.004434 0.005703 −0.001288 0.119524 0.006504 0.007270 Fndc3c1 −0.000850 −0.005531 0.006357 0.012678 −0.002435 −0.001269 Fndc5 0.013699 0.007673 −0.021619 0.045342 0.013837 −0.003374 Foxn2 0.040966 −0.015444 −0.026432 −0.001597 −0.018413 −0.006774 Frem3 0.006236 −0.004570 −0.001688 0.002182 0.012172 0.042928 Fuk −0.007673 −0.001861 0.009481 0.018893 0.028899 0.028713 Fut9 0.037894 −0.018821 −0.019834 −0.034702 −0.014858 −0.029450 Gabra2 −0.012137 0.020750 −0.008840 0.038159 0.027390 0.032088 Gabra4 0.027922 −0.033223 0.004643 −0.031251 −0.057597 −0.027506 Gabrg1 −0.022641 −0.006214 0.028384 0.016926 0.030820 0.053906 Gabrg2 −0.006649 −0.000634 0.007249 0.017533 0.031502 0.029195 Gale −0.007989 0.014799 −0.006925 −0.009497 0.017537 0.006901 Galntl6 0.003718 −0.000059 −0.003669 0.034606 0.010471 0.015599 Gas2l1 −0.006452 0.003168 0.003262 0.021277 0.030483 −0.008577 Gcc1 0.007802 −0.013064 0.005172 0.023867 0.030108 0.025183 Gcnt1 −0.023484 0.001719 0.021414 −0.021974 −0.031170 −0.041239 Gemin4 −0.016717 0.005171 0.011395 0.036510 0.027933 0.024970 Glg1 0.008494 −0.010990 0.002427 0.037385 0.000554 0.010301 Gli1 −0.005896 −0.002743 0.008599 0.023753 0.017393 0.054424 Glis3 −0.013147 0.039712 −0.027435 0.016750 0.017155 0.018998 Glra2 −0.016243 −0.007444 0.023386 0.051520 0.006376 0.004759 Gm11346 0.000188 0.008639 −0.008881 0.032643 0.007474 −0.002293 Gm11974 −0.011616 −0.005927 0.017378 0.025509 0.020509 −0.015357 Gm14164 −0.011594 0.012996 −0.001507 0.022174 0.023665 0.019001 Gm15880 0.005391 0.010988 −0.016525 −0.027724 −0.027951 −0.030568 Gm16740 0.001744 −0.005880 0.004117 0.015317 0.012325 0.021215 Gm17750 −0.030053 0.011486 0.018096 0.061282 0.042948 0.016125 Gm17769 −0.002742 −0.004903 0.007614 0.014283 0.019365 0.019778 Gm20324 −0.003894 0.000389 0.003495 0.031336 0.024251 0.037550 Gm6981 0.003595 −0.003087 −0.000515 0.009235 0.021544 0.013821 Gm7173 0.023755 −0.003202 −0.020903 −0.034955 −0.032778 −0.024608 Gpatch1 0.009433 −0.006669 −0.002813 0.276563 −0.004951 0.271455 Gpm6a 0.029635 −0.017120 −0.012981 −0.021001 −0.014083 −0.030517 Gpr137 −0.003506 0.016493 −0.013146 0.028135 0.054863 0.017631 Gps2 −0.004506 0.003335 0.001160 0.009569 0.006660 0.012407 Grhl3 −0.026228 0.013782 0.012092 0.018489 0.036071 0.048555 Gria1 0.022711 −0.017778 −0.005231 −0.027065 −0.034088 −0.027728 Grik4 −0.007499 0.015619 −0.008249 0.018959 0.011380 0.004104 Grin1 −0.002179 0.013335 −0.011264 0.006259 0.074773 0.024808 Gsr −0.000673 −0.000140 0.000813 0.004357 0.034764 0.025348 Gsta4 −0.018132 0.007389 0.010572 0.027919 0.025005 0.033530 Gstm6 −0.005456 −0.001919 0.007345 0.028025 0.030047 0.023084 Gtf3c3 0.011578 −0.002246 −0.009411 0.018668 0.009081 −0.005064 Gucy1b3 0.016377 0.008318 −0.025028 −0.028804 −0.016967 −0.031624 Hcn4 −0.018446 0.011088 0.007180 0.017274 0.017466 0.051995 Hdgf −0.000720 −0.009349 0.010004 0.028164 0.023238 0.010883 Hebp2 −0.011866 −0.004097 0.015822 −0.022133 −0.011312 −0.029528 Heca 0.017557 −0.001909 −0.015842 0.038117 0.021492 0.009732 Hist1h2bg 0.000117 −0.005956 0.005815 0.002649 0.006984 0.043465 Hist1h4b −0.017282 −0.004497 0.021508 0.024550 0.021334 0.042603 Hist2h3b −0.013489 −0.005956 0.019241 0.031055 0.060190 0.007006 Hmgb1 0.024149 −0.018646 −0.005838 −0.013967 0.003214 −0.000944 Hnrnph2 0.010933 −0.003525 −0.007472 −0.007997 −0.006749 −0.016987 Hpca −0.002052 0.019062 −0.017241 −0.027830 −0.022917 −0.051506 Hs6st2 0.021908 −0.003338 −0.018863 −0.032517 −0.017529 −0.029308 Hsd3b6 0.004440 −0.006236 0.001774 0.010785 0.013232 0.015326 Htr2c 0.000032 0.001532 −0.001566 0.027368 0.047326 0.034566 ICa1 0.004933 0.008384 −0.013412 −0.030380 −0.018243 −0.049227 Il1rap 0.003743 −0.001886 −0.001865 0.028159 0.025304 0.029632 Inpp5f 0.017837 −0.001651 −0.016390 −0.016983 −0.021727 −0.019904 Iqcb1 0.019984 −0.001183 −0.019065 −0.034960 −0.009793 −0.004832 Irs1 −0.002404 −0.004789 0.007164 0.017327 0.010217 0.023965 Irs4 0.003252 0.021427 −0.025059 0.048015 0.049916 0.028053 Irx2 −0.011814 0.010835 0.000889 0.034875 0.024138 0.018274 Ism1 −0.002012 0.003227 −0.001221 0.028359 0.017715 0.024066 Itga1 0.006588 −0.019118 0.012336 −0.029112 −0.004857 −0.025854 Jak2 0.022039 −0.010863 −0.011432 −0.014345 −0.017060 −0.030606 Kank2 −0.031797 0.030973 0.000141 0.054543 0.033444 0.018352 Kcnd1 0.003423 −0.002401 −0.001028 0.005455 0.014243 0.036797 Kcnh2 −0.015605 0.000170 0.015270 0.030850 0.028812 0.043416 Kctd5 −0.018456 −0.005896 0.024021 0.026000 0.026669 0.037530 Kdm1b 0.023451 −0.025446 0.001580 −0.037429 −0.014961 −0.033708 Khk −0.000828 0.000802 0.000026 0.023286 0.028787 0.028469 Kif27 −0.007329 −0.000466 0.007756 0.030706 0.017263 0.010164 Kiss1r −0.014366 0.005794 0.008464 0.017392 0.014083 0.026831 Kitl 0.043310 −0.028800 −0.015535 −0.015555 −0.035147 −0.038968 Kmt2d 0.022014 −0.037309 0.014575 −0.011650 −0.013011 0.031589 Knstrn −0.003808 −0.012292 0.015954 −0.030941 −0.016816 −0.020409 Kri1 −0.009467 0.000410 0.008997 0.042767 0.024702 0.004916 Lama5 −0.012741 −0.000298 0.012925 0.042904 0.024527 0.022320 Lamtor5 0.023062 −0.010714 −0.012627 −0.029369 −0.015333 −0.018669 Lars2 −0.009459 0.000777 0.008625 0.035127 0.015773 0.014660 Lbh −0.009505 0.005817 0.003640 0.007157 0.008740 0.022590 Lingo1 0.002140 0.011866 −0.014125 0.009461 0.017063 0.017708 Lipt2 0.010499 0.009588 −0.020299 −0.018888 −0.028080 −0.035831 Lmx1b −0.017149 0.032330 −0.015732 −0.020461 −0.023881 −0.036729 LOC100862268 0.009827 −0.002010 −0.007874 −0.044395 −0.032873 −0.014472 Loxl2 −0.016265 0.043373 −0.028131 0.085802 0.036707 0.017138 Lpcat1 −0.006534 0.003221 0.003291 0.001024 0.010501 0.002687 Lppr4 −0.005162 0.004518 0.000627 0.041989 0.029693 0.018026 Lrfn1 −0.009936 0.020630 −0.010918 0.052619 0.028086 −0.014969 Lrp1b 0.026165 −0.004242 −0.022340 0.021101 0.023812 0.003189 Lrp5 −0.018291 0.007110 0.011006 0.043552 0.054254 −0.003701 Lrr1 −0.021191 0.002217 0.018696 0.020177 0.020899 0.022818 Lrrc29 0.005285 −0.011797 0.006439 0.041038 −0.007727 0.003862 Lsm12 0.000810 −0.005931 0.005100 −0.005960 0.027091 0.003818 Magel2 −0.003451 −0.002270 0.005704 0.028603 0.011827 0.017125 Map1a −0.020664 0.001537 0.018856 0.021269 0.028047 0.035120 Map3k14 −0.032854 0.006272 0.025962 −0.012985 0.026582 −0.001720 Map3k19 −0.008404 −0.004815 0.013126 0.057283 0.020289 0.022257 Map3k5 −0.081534 0.147625 −0.078253 0.053402 0.108373 −0.079263 Map7d1 −0.018393 0.004044 0.014156 0.023904 0.022336 0.016662 Mapk8 0.017699 −0.014199 −0.003683 −0.016678 −0.025807 −0.023579 Mapkapk2 −0.019839 0.009033 0.010602 0.028023 0.016046 0.036993 Maz −0.016298 −0.001251 0.017351 0.015865 0.024987 0.027710 Mcc −0.014304 0.000542 0.013627 0.067357 0.007270 0.021967 Mcfd2 0.000426 0.009580 −0.010072 −0.002821 −0.020723 −0.020233 Mdc1 −0.030340 0.001336 0.028405 0.048642 0.041175 0.034226 Med10 0.009287 −0.008403 −0.000939 −0.022674 −0.021754 −0.024607 Mier2 −0.008238 0.008359 −0.000169 0.012308 0.019737 0.053714 Mier3 0.020612 −0.000336 −0.020570 −0.013814 −0.019443 −0.029076 Mir3081 0.000351 −0.009182 0.008775 0.010811 0.005576 0.035663 Mir411 0.006437 0.003800 −0.010294 0.019085 0.013746 0.025397 Mir6236 −0.013751 0.005090 0.008561 0.076410 0.032753 −0.000536 Mir6353 0.003664 −0.002011 −0.001660 −0.005129 0.046421 0.037742 Mllt6 0.011340 −0.008914 −0.002500 0.005730 0.020099 0.026116 Mpeg1 −0.024779 0.038005 −0.014010 0.011250 0.034966 0.025008 Mpnd 0.010233 0.007810 −0.018215 −0.013031 −0.032260 −0.013999 Mrpl22 0.009821 −0.002185 −0.007692 −0.004029 −0.016490 −0.021003 Mrps18b −0.002930 −0.007410 0.010281 −0.016610 −0.029240 −0.028656 Mrps33 0.012591 −0.011405 −0.001286 −0.019534 −0.015692 −0.002915 Mtcl1 −0.017168 0.013297 0.003703 0.020775 0.022441 0.021541 Mttp 0.002595 0.003111 −0.005723 −0.017351 −0.019059 −0.046066 Mtx3 0.013996 −0.007548 −0.006551 −0.010210 −0.011360 −0.014171 Mum1 0.007587 0.006498 −0.014189 0.009866 −0.003450 −0.009214 Mvk −0.000333 −0.003340 0.003664 −0.001120 0.008702 0.005543 Myh14 −0.008945 0.002133 0.006766 0.082437 0.007931 0.003062 Myo5b −0.004927 0.011230 −0.006369 0.048034 0.001301 −0.006029 Myo6 −0.007993 0.003364 0.004596 0.021841 −0.007853 −0.022369 Mzf1 −0.017218 0.001290 0.015739 −0.018263 −0.023176 −0.011228 Nans 0.000711 0.001281 −0.001995 −0.034288 0.001118 −0.018664 Nap1l5 0.006232 −0.008835 0.002560 −0.033905 −0.043003 −0.046929 Ncmap −0.003776 0.015359 −0.011718 0.025284 0.005807 0.024104 Ncor2 −0.015291 0.028574 −0.013713 −0.024608 −0.030869 −0.007514 Ndufs4 0.011829 −0.009173 −0.002736 −0.031533 −0.020066 −0.015932 Necab1 0.008373 −0.000349 −0.008070 −0.031710 −0.029761 −0.041638 Nek11 −0.002918 −0.011713 0.014507 −0.007641 −0.025728 −0.009841 Nexn 0.028425 −0.004457 −0.024463 −0.018251 −0.010557 −0.010331 Nfs1 −0.002135 −0.006732 0.008823 −0.034498 −0.025832 −0.026292 Nipa2 0.013274 −0.015719 0.002297 −0.039300 −0.012938 −0.031812 Nipal2 −0.014130 0.005121 0.008903 −0.020718 −0.034423 −0.030661 Nmd3 0.017927 −0.005239 −0.012867 −0.020403 −0.024094 −0.037752 Nol4 0.016709 −0.018429 0.001505 −0.041465 −0.012868 −0.014388 Notch3 −0.040705 0.008761 0.031013 0.028291 0.041644 0.044335 Nptxr −0.003279 0.006671 −0.003415 0.031615 0.017866 0.024378 Npy1r 0.001244 0.011105 −0.012445 0.037032 0.018236 −0.030671 Nr5a2 0.009878 0.005378 −0.015382 −0.004256 −0.006021 0.002077 Nrcam 0.012295 −0.006825 −0.005549 0.009287 0.011469 0.003472 Nsun6 0.020092 −0.009873 −0.010431 −0.030156 −0.021543 −0.029621 Ntn1 0.005978 −0.001328 −0.004671 −0.028408 −0.019801 −0.035034 Ntng1 0.021517 0.009888 −0.031951 −0.029923 −0.039840 −0.032436 Nudt11 −0.004422 −0.014007 0.018239 0.007596 −0.004717 −0.003711 Nup214 0.017379 0.013747 −0.031641 −0.041918 −0.047491 −0.043206 Oxsm 0.004297 −0.011414 0.007048 0.001348 0.024905 0.025040 Pcdh20 0.023639 −0.001203 −0.022811 −0.026525 −0.038849 −0.038645 Pcdh7 0.003534 0.011239 −0.014898 −0.010953 −0.019754 −0.035595 Pcdha11 −0.022670 0.006607 0.015785 −0.010027 −0.044659 −0.044411 Pcdha7-g −0.010005 −0.006479 0.016342 −0.034866 −0.006901 −0.025434 Pcdhb12 −0.012498 0.006894 0.005523 −0.027142 −0.044063 −0.026985 Pcdhb20 0.018064 0.000261 −0.018557 −0.025144 −0.023118 −0.031505 Pcdhb22 −0.011889 0.008329 0.003484 −0.035698 −0.027813 −0.037372 Pcdhgb5 −0.003894 −0.009070 0.012872 −0.039541 −0.035397 −0.050953 Pde4d −0.006608 0.012885 −0.006364 −0.044989 −0.031616 −0.035750 Pdia4 −0.006346 0.001444 0.004878 0.033812 0.031569 0.002745 Pdss2 0.004271 0.007096 −0.011436 0.029407 0.016528 0.035478 Pex11b 0.001524 −0.000007 −0.001519 −0.035175 −0.028986 −0.013867 Pgbd1 0.004859 0.000938 −0.005818 −0.022896 0.001550 −0.011179 Phf5a 0.001928 −0.016349 0.014257 −0.031943 −0.026539 0.000463 Phyhip 0.008792 0.006281 −0.015192 −0.017888 −0.036329 −0.038415 Pip4k2a 0.010530 −0.002978 −0.007615 −0.013783 −0.020200 −0.030017 Pisd-ps3 −0.045314 0.010817 0.033364 −0.003239 −0.038375 −0.055206 Plagl2 0.006606 0.005413 −0.012095 −0.017949 −0.019388 −0.048270 Plcd −0.007514 0.019600 −0.012291 0.031706 −0.000887 0.018776 Pld2 0.008658 0.005440 −0.014205 −0.037315 −0.041158 −0.016724 Plek 0.023422 0.012021 −0.036133 −0.020604 −0.009310 −0.016587 Plekhn1 −0.018748 −0.000333 0.018836 0.010464 0.045068 0.034149 Plp2 0.003632 0.000865 −0.004509 −0.039208 −0.068040 −0.034770 Plxna3 0.010246 0.003164 −0.013512 −0.020572 −0.022906 −0.029430 Pmaip1 −0.015985 0.012651 0.003187 −0.033896 −0.040987 −0.031887 Poli −0.008654 −0.003660 0.012231 −0.004677 −0.054700 −0.033878 Polr3c 0.001293 −0.004817 0.003512 −0.008154 −0.017295 0.011259 Polr3gl −0.016605 −0.000946 0.017351 0.011359 0.014296 0.008375 Polrmt −0.008069 0.010393 −0.002386 0.032318 0.002574 0.025351 Pou2f2 0.007522 −0.004407 −0.003144 0.027385 0.023020 0.044023 Pou6f2 0.016915 −0.003890 −0.013190 −0.038737 −0.031669 −0.004319 Ppap2b 0.003073 −0.006233 0.003140 −0.027927 −0.031911 −0.038361 Ppp1r3fos 0.025256 0.005432 −0.031256 0.011713 −0.016233 0.018778 Prr12 0.019562 0.007179 −0.027145 −0.038234 −0.023189 −0.032791 Prr36 0.010415 −0.017502 0.006927 0.013732 −0.000494 0.039650 Prrc2a −0.022121 −0.014583 0.036009 0.017364 0.031456 0.041011 Prrg1 0.024562 −0.009391 −0.015495 −0.030528 −0.066212 −0.030298 Psg16 0.010298 −0.002768 −0.007589 −0.030109 −0.021847 −0.067258 Pstpip2 −0.014425 0.015278 −0.001007 −0.018167 −0.028999 −0.029915 Ptch2 −0.010309 0.007732 0.002517 0.032688 0.030106 0.024728 Ptk2b −0.022265 0.013349 0.008657 −0.055288 −0.024587 −0.035177 Ptprf −0.024287 0.002195 0.021723 0.020469 0.022752 0.024853 Pus7 −0.016915 0.000304 0.016418 −0.018272 −0.034009 −0.034447 Pxn 0.011143 −0.006321 −0.004887 −0.018441 0.002311 0.002613 Rapgef1 −0.006112 −0.015870 0.021718 0.022698 0.017193 0.035272 Rcan2 −0.016526 0.020618 −0.004340 −0.023357 −0.041106 −0.021282 Reps2 0.022723 −0.007819 −0.015185 −0.039149 −0.031480 −0.041976 Rgs17 0.012416 −0.005070 −0.007428 −0.028242 −0.043635 −0.022940 Rhof 0.005288 −0.012451 0.007082 −0.030062 −0.034903 −0.025996 Ric8b 0.015228 −0.007809 −0.007541 −0.014912 −0.034270 −0.020248 Rilpl1 0.002712 0.003608 −0.006341 −0.005501 −0.007587 −0.009483 Rin3 −0.014830 0.004207 0.010502 0.016494 0.034052 0.035408 Rint1 0.010160 −0.003744 −0.006471 −0.038676 −0.050862 −0.006302 Rn45s 0.012530 0.006240 −0.018962 −0.034615 −0.051512 −0.018833 Rnf126 −0.028653 0.014928 0.013304 0.013297 0.022822 0.023288 Rnpep −0.007857 −0.000687 0.008498 0.034274 0.014885 0.027546 Rnpepl1 −0.019349 −0.001627 0.020697 0.040673 0.015002 0.031729 Rpl19 −0.009816 0.005865 0.003901 −0.003314 −0.026377 −0.009122 Rpl23a −0.007372 −0.011625 0.018808 −0.010686 0.007123 0.001361 Rpl3 −0.007286 0.015932 −0.008780 −0.002991 −0.023046 −0.018411 Rpn2 −0.012772 0.012127 0.000538 0.040068 0.020043 0.007737 Rps12 −0.006454 0.012701 −0.006331 −0.000015 −0.018088 −0.004917 Rps2 −0.003782 −0.002971 0.006730 −0.003623 −0.009712 0.008957 Rwdd2b 0.010165 −0.003971 −0.006249 −0.010412 −0.037133 −0.065397 Rxfp3 −0.012760 0.021961 −0.009457 0.036721 0.013645 0.025178 Ryr2 0.009468 0.012583 −0.022309 −0.038208 −0.022667 −0.030954 S100a11 0.002840 0.013107 −0.016099 −0.006264 0.004612 −0.019450 Sall2 −0.011825 0.002894 0.008853 0.016678 0.017757 0.025393 Sarnp 0.019599 −0.029818 0.009746 −0.014753 0.004823 −0.021101 Sash3 0.000140 −0.019687 0.019285 0.000651 −0.035287 −0.030888 Scaf1 −0.024879 0.010482 0.014076 0.024125 0.031139 0.036283 Scd4 0.000404 −0.001321 0.000916 0.039579 0.005706 0.029497 Scn1a 0.029830 −0.000974 −0.029465 −0.030302 −0.026805 −0.066524 Scrib −0.019934 0.010178 0.009551 0.014896 0.026881 0.030888 Scrt1 0.001305 0.010579 −0.011973 −0.022429 −0.024621 −0.024599 Scube1 −0.025287 0.029126 −0.004362 −0.031709 −0.034672 −0.038635 Scx −0.012935 0.024398 −0.011776 −0.013270 −0.029412 −0.027498 Sdha 0.004352 0.005067 −0.009465 −0.002049 −0.011239 −0.007797 Sema3f 0.001632 −0.004266 0.002624 −0.025954 −0.024357 −0.019785 Sema6a 0.012876 0.024367 −0.038004 −0.060562 −0.022630 −0.065122 Senp7 0.019679 −0.011813 −0.008072 −0.018142 −0.014763 −0.039642 Sfxn4 0.007113 −0.000770 −0.006375 −0.030690 −0.025937 −0.022274 Sgk3 −0.002299 −0.019698 0.021697 −0.057281 −0.023063 −0.044107 Sh3bgrl2 −0.003420 0.013913 −0.010603 −0.017819 −0.026839 −0.025606 Sh3bp4 −0.008671 −0.002980 0.011576 0.033888 0.030763 0.018081 Shisa9 0.008869 0.008787 −0.017819 −0.039235 −0.030927 −0.023212 Shmt2 −0.011753 −0.026609 0.037577 0.039428 0.032039 0.030435 Sigmar1 0.004379 −0.003622 −0.000769 0.022653 0.023540 0.038625 Slc10a3 0.004188 −0.008621 0.004395 0.038554 0.020932 0.031304 Slc15a4 0.011103 −0.003419 −0.007752 −0.037626 −0.019966 −0.058918 Slc17a5 0.019953 −0.017586 −0.002615 −0.035722 −0.024544 −0.025056 Slc1a5 −0.013425 0.006442 0.006890 −0.035908 −0.038050 −0.017909 Slc22a12 −0.014598 0.006600 0.007887 0.001005 −0.016955 −0.002747 Slc24a3 −0.006701 −0.011528 0.018054 −0.043770 −0.022563 −0.030934 Slc25a1 −0.017147 0.009831 0.007163 0.014053 0.032417 0.020103 Slc29a4 0.016903 −0.016003 −0.001088 0.033645 0.017912 0.032389 Slc35f3 −0.015911 0.019353 −0.003664 0.001338 −0.023620 −0.000806 Slc36a4 0.019354 0.014566 −0.034534 −0.035845 −0.010997 −0.050029 Slc37a2 −0.010758 −0.007981 0.018557 0.020876 0.027231 0.031998 Slc52a2 −0.013902 0.025494 −0.011934 0.013856 0.011449 0.033675 Slc6a8 −0.003339 −0.001963 0.005286 0.018356 0.030737 0.018864 Slco2b1 −0.010552 0.011054 −0.000583 0.043834 0.048050 0.014267 Slitrk4 0.007124 0.014255 −0.021628 −0.022324 −0.033070 −0.034216 Smarca5-ps 0.005849 0.009971 −0.015954 −0.037944 −0.027329 −0.024792 Smim20 −0.022286 0.005181 0.016826 −0.028832 −0.042365 −0.016469 Smim3 −0.010199 0.006428 0.003716 0.052992 0.021058 0.012297 Smim8 −0.019799 0.019320 0.000213 −0.007489 −0.007999 −0.011845 Smpdl3a −0.026893 −0.013970 0.039987 −0.024862 −0.022771 −0.024425 Snhg1 −0.003079 −0.011730 0.014683 −0.029954 −0.015735 0.001372 Snora15 0.000551 −0.013847 0.013169 −0.038679 −0.026570 −0.037862 Snora23 0.021673 −0.001825 −0.020153 −0.012766 −0.021076 −0.025328 Snord91a 0.002435 −0.005583 0.003132 0.011095 0.044474 0.020983 Socs6 −0.000791 0.026411 −0.026097 −0.027661 −0.030910 −0.019523 Sox13 0.000539 0.005293 −0.005853 0.025914 0.029097 0.030091 Sox4 −0.001663 −0.004362 0.006005 0.000642 0.006085 0.007327 Spry3 0.010023 0.013463 −0.023778 0.000726 0.016737 −0.000700 Spsb1 −0.000132 0.013928 −0.013930 −0.021138 −0.020941 −0.005620 Sqrdl −0.004965 0.003341 0.001611 −0.017190 −0.027351 −0.028406 Srbd1 0.000813 −0.000241 −0.000572 −0.020799 0.011609 −0.020928 Src −0.021455 0.005947 0.015256 0.017420 0.028482 0.024690 Srcap 0.002458 −0.015759 0.013153 0.014770 0.014343 0.026788 Srp54b 0.001813 0.008845 −0.010726 −0.024740 −0.020908 −0.005128 St3gal3 −0.001947 0.013125 −0.011283 0.023552 0.019318 0.034874 St8sia6 0.043534 −0.024469 −0.020073 −0.043456 −0.041574 −0.026120 Stard5 −0.005244 −0.006021 0.011199 −0.018216 −0.025443 −0.034054 Stxbp3-ps 0.011052 0.009172 −0.020440 −0.037943 −0.013422 −0.040494 Supt6 0.003228 −0.005452 0.002208 0.014245 0.014766 0.024974 Sv2b 0.038616 −0.001189 −0.038457 −0.042965 −0.049963 −0.040797 Svopl −0.015065 0.015237 −0.000331 −0.006704 0.002560 −0.012317 Synpo2 −0.023778 0.012197 0.011290 0.027852 0.057355 0.010588 Syt17 0.013027 −0.003593 −0.009529 −0.036883 −0.014745 −0.025348 Syt3 −0.008239 0.009751 −0.001570 −0.020546 −0.036464 −0.028218 Syvn1 −0.019328 0.009312 0.009824 0.011102 0.037692 0.039538 Tacc1 −0.000906 0.005976 −0.005092 −0.021800 0.021032 −0.026515 Tada1 0.001590 −0.003640 0.002043 −0.034684 −0.008010 −0.027514 Taf1a 0.017011 −0.005710 −0.011459 −0.020314 −0.009568 −0.020910 Taf4a 0.031887 0.009970 −0.042877 −0.033405 −0.042192 −0.044984 Taok2 0.004774 0.000322 −0.005113 0.017756 0.021997 0.014852 Tas1r1 0.001524 −0.001561 0.000035 −0.015380 −0.008555 −0.003584 Tatdn1 −0.025233 0.020336 0.004527 −0.031775 −0.030837 −0.031760 Tbc1d10a −0.008675 0.001772 0.006860 0.022187 −0.001686 0.006209 Tbc1d4 −0.009719 0.003963 0.005707 −0.019637 0.001366 −0.005345 Tbc1d9 −0.004999 0.005437 −0.000457 −0.001518 0.017596 0.007560 Tbx2 −0.029950 0.019117 0.010360 0.081200 0.023088 0.017227 Tcf3 −0.030448 −0.003033 0.032783 0.022485 0.023775 0.043199 Tenm1 0.010046 0.015652 −0.026052 −0.023777 −0.035682 −0.030103 Tfcp2l1 0.020540 −0.001251 −0.019568 −0.036448 −0.034980 −0.037405 Them4 0.002209 0.014996 −0.017390 −0.025831 −0.014387 −0.016045 Thoc7 0.016809 −0.017870 0.000852 −0.036856 −0.027174 −0.034740 Thsd4 0.006019 0.005836 −0.011929 −0.047726 −0.046408 −0.006245 Tigar −0.002701 0.011342 −0.008714 0.017069 0.037864 0.033421 Timm9 −0.005605 0.013310 −0.007798 −0.004740 −0.000800 0.012257 Tm4sf1 0.028563 −0.006535 −0.022502 −0.015783 −0.005930 −0.017539 Tmc7 −0.006083 0.004833 0.001228 −0.011373 0.004561 −0.031664 Tmem170b −0.000658 −0.004610 0.005251 −0.001248 0.008376 −0.007683 Tmem180 −0.003636 −0.001435 0.005057 −0.042603 −0.028332 −0.022672 Tmem185b 0.011836 −0.014195 0.002240 −0.001541 −0.004751 −0.012033 Tmem203 −0.012320 0.001317 0.010908 −0.022027 −0.025258 −0.015142 Tmem29 0.005702 −0.007076 0.001345 −0.014522 −0.030633 −0.053734 Tmem81 −0.021138 −0.000753 0.021574 −0.020405 −0.030635 −0.031197 Tmem8b −0.017438 0.006082 0.011195 0.026036 0.020533 0.009567 Tmem9 0.009546 −0.004700 −0.004894 −0.028288 −0.032638 −0.018057 Tmppe −0.016695 −0.004086 0.020531 0.021167 0.022400 0.031954 Tnfrsf19 0.006787 −0.010305 0.003462 0.008369 0.027508 0.031933 Tomm5 0.000929 0.012976 −0.014031 −0.017698 −0.038827 −0.037850 Tpcn1 −0.011163 0.008714 0.002378 0.009310 0.040114 0.049309 Trim13 0.019233 −0.004542 −0.014903 −0.029189 −0.013496 −0.030691 Trim9 0.007306 −0.006910 −0.000431 −0.006094 −0.006103 −0.001308 Tshz3 0.002918 0.015158 −0.018274 −0.048314 −0.012300 −0.039350 Tspan13 0.007890 −0.000036 −0.007897 −0.006777 −0.009938 −0.012938 Ttc39aos1 −0.003346 0.009217 −0.005917 −0.023948 −0.012736 −0.006059 Tuft1 0.001296 0.001697 −0.002998 0.003914 −0.008725 0.001321 Txnrd3 0.003943 −0.004030 0.000075 −0.040823 −0.029196 −0.022389 Ubap2 0.007254 −0.021887 0.014378 0.011429 0.026834 0.034243 Ubb 0.005381 −0.007630 0.002217 −0.023365 −0.020315 −0.010179 Ube2j2 0.008102 −0.008789 0.000637 0.010031 −0.007326 −0.011033 Ube2r2 −0.003064 −0.004666 0.007698 0.021720 0.012959 0.021269 Uck2 0.001256 −0.007776 0.006484 0.023998 0.030929 0.022775 Utp11l 0.003668 0.007234 −0.010967 −0.016373 −0.014688 −0.019780 Vac14 0.009450 −0.014758 0.005192 0.004690 0.010709 0.039612 Vamp7 −0.007581 0.009221 −0.001691 −0.048348 −0.037779 −0.013205 Vangl2 −0.008416 −0.013115 0.021290 0.016466 0.019586 0.050076 Vasp −0.026147 0.013322 0.012474 0.032668 0.028802 0.036944 Vcpip1 0.021563 −0.002627 −0.019228 −0.021589 −0.015551 −0.022061 Vwa8 −0.001728 0.008831 −0.007148 0.028282 0.021707 0.021273 Vwc2l −0.009752 0.011764 −0.002095 −0.027229 −0.022005 −0.026290 Wfs1 0.017082 0.008069 −0.025499 −0.028008 −0.024851 −0.042632 Xlr3a −0.000673 0.002167 −0.001497 −0.029158 −0.021372 −0.030573 Xylt1 0.007624 0.025160 −0.033409 −0.031928 −0.042862 −0.034861 Yipf2 0.016655 −0.004826 −0.011983 0.011403 −0.006338 0.034880 Zbtb45 0.018195 −0.004690 −0.013692 −0.036749 −0.026237 −0.029094 Zbtb46 0.023025 0.001658 −0.025086 −0.023924 −0.033067 −0.041451 Zc3h10 0.011316 −0.015060 0.003617 −0.008523 −0.023412 −0.002397 Zc3h12b 0.008060 0.015084 −0.023435 −0.029989 −0.037946 −0.030011 Zc3h18 −0.014044 −0.004449 0.018301 0.014335 0.028638 0.029885 Zeb2os 0.011561 −0.004064 −0.007569 −0.040244 −0.029679 −0.027164 Zfhx3 0.016317 −0.003908 −0.012561 −0.031074 −0.014626 −0.011462 Zfp212 −0.004695 −0.000668 0.005345 −0.005263 0.004276 0.009323 Zfp330 0.006770 −0.006066 −0.000733 −0.018707 −0.020203 −0.030827 Zfp35 0.020093 0.002335 −0.022749 −0.038052 −0.015884 −0.041656 Zfp362 0.002379 −0.011557 0.009102 0.035827 0.021264 0.019315 Zfp36l1 0.010031 −0.020700 0.010448 0.035114 0.041138 0.016446 Zfp628 0.018878 −0.012513 −0.006558 0.005431 −0.020178 0.001984 Zfp651 −0.007410 0.010234 −0.002882 0.023355 0.014911 0.014142 Zfp710 0.000379 −0.013536 0.013035 0.017175 0.033010 0.025302 Zfp809 −0.013000 0.011480 0.001416 0.003336 −0.001140 −0.008168 Zfp839 −0.002056 0.000260 0.001793 0.006397 0.014099 0.007840 Zfp85 0.014042 0.018837 −0.033456 −0.021231 −0.046299 −0.031123 Zfp850 0.010219 −0.008903 −0.001380 0.006155 −0.004383 −0.006469 Zfpm1 −0.021619 −0.001184 0.022465 0.000153 0.020359 −0.023700 Zic5 −0.011661 0.014379 −0.002840 −0.037437 −0.021909 −0.049901 Zmynd10 −0.015754 −0.000094 0.015677 −0.025473 −0.032960 −0.029013 Zscan12 0.004344 −0.016879 0.012376 −0.016942 −0.015238 −0.011926 Zscan2 −0.004701 0.013244 −0.008638 0.014394 0.032285 0.032744 Zswim8 −0.017699 0.000092 0.017393 0.015412 0.021578 0.035259

TABLE 1B gene SP1 SP2 SP3 SPF v ABX p-value Sp v ABX p-value 1700017B05Rik 0.018638 −0.004432 0.049365 0.020742 0.346030 1810055G02Rik 0.031917 0.020735 −0.014757 0.003495 0.971940 2610015P09Rik 0.018861 −0.000999 0.032038 0.296571 0.025047 2610020C07Rik 0.035140 0.036800 −0.012333 0.992996 0.011782 2700046A07Rik −0.001661 −0.007484 −0.002094 0.013359 0.116251 2810408I11Rik 0.004373 0.009511 0.020696 0.295913 0.044638 2810468N07Rik 0.014534 0.026114 0.051083 0.383007 0.015117 3830406C13Rik 0.020414 0.064631 0.034712 0.927670 0.005845 4930447A16Rik −0.028895 −0.028465 −0.018808 0.049218 0.914526 4930511A08Rik 0.011405 0.005374 0.003392 0.781974 0.036947 4933439C10Rik −0.007199 −0.023143 −0.006832 0.021055 0.166271 5930403L14Rik 0.037099 0.001931 −0.005865 0.047893 0.233449 6330415G19Rik −0.011281 −0.015162 −0.007935 0.027276 0.187974 6330549D23Rik −0.018105 −0.024910 −0.055949 0.002299 0.030601 6430573F11Rik 0.031887 0.018290 0.038128 0.014222 0.629827 6530402F18Rik 0.006070 −0.017917 −0.021293 0.209688 0.043199 9130019P16Rik −0.002109 −0.019938 −0.001767 0.136514 0.041045 9430065F17Rik 0.011807 0.018180 0.003938 0.008398 0.095445 A330069E16Rik −0.031137 −0.015269 −0.018878 0.023845 0.535954 A930003O13Rik −0.003694 −0.002218 −0.000522 0.063207 0.040377 Acad9 −0.020673 −0.013996 −0.036059 0.964761 0.003238 Acsbg1 −0.009494 −0.002390 −0.024497 0.195912 0.032880 Acsf2 0.007830 0.021217 0.002728 0.715813 0.026933 Adamts8 −0.005202 −0.008857 −0.014714 0.008054 0.365332 Agap3 0.003172 −0.004897 −0.009828 0.036757 0.180482 Airn −0.032133 0.008081 −0.013357 0.212826 0.044750 Ak7 0.030155 0.012369 0.058683 0.113220 0.045880 Aldoc 0.019091 0.005108 −0.009872 0.167400 0.036699 Alpl −0.018661 −0.005526 −0.019086 0.819387 0.014538 Ankrd11 0.007595 0.014692 0.002831 0.897735 0.004728 Ankrd6 0.018971 0.010460 0.002472 0.187595 0.028505 Ap1s1 0.011447 0.022753 0.017971 0.041310 0.298544 Ap3s1 −0.015266 0.013899 0.004645 0.039435 0.292725 Arhgap10 0.018213 0.023605 −0.012254 0.329280 0.020090 Arid1a 0.014766 0.016211 −0.004863 0.039376 0.036938 Arl6ip1 −0.014357 −0.012329 −0.038201 0.466523 0.016309 Armt1 0.007545 0.015379 0.022269 0.032507 0.176439 Arrb2 0.007874 0.024731 0.023994 0.020977 0.008638 Arsa −0.028240 −0.004958 −0.037616 0.334779 0.034061 Arxes2 0.022150 0.034058 0.015139 0.028753 0.562367 Ate1 −0.016000 −0.011618 −0.026385 0.590575 0.028250 Atg4d −0.022400 −0.003575 −0.023475 0.581787 0.042946 Atn1 −0.039266 −0.019820 −0.026779 0.028445 0.762889 Atp5h 0.006702 −0.002794 0.005798 0.414676 0.041331 Atp6ap2 0.001933 −0.018617 0.000114 0.001091 0.018647 Atp6v1b2 −0.008792 −0.012530 −0.025810 0.069472 0.029589 Atp8b1 0.003365 0.013959 0.000712 0.034991 0.104766 Atpaf1 0.010982 0.016225 0.009383 0.026162 0.675041 Atxn2 0.021957 0.020479 0.019710 0.036812 0.110005 Aven 0.004074 0.016877 0.016698 0.028744 0.212447 AW209491 0.003241 0.022375 0.015654 0.010607 0.424536 AW549877 −0.000175 0.002798 0.031642 0.036804 0.255979 Axin2 0.010689 −0.004838 0.018189 0.015686 0.104696 B230307C23Rik −0.012088 −0.017021 −0.034730 0.559672 0.037047 B3galt2 −0.037332 −0.008679 −0.009353 0.048749 0.560575 B4galt4 0.007768 0.027834 −0.007646 0.129087 0.029637 Bahcc1 0.025570 0.013260 0.018734 0.025823 0.744577 Baiap2l1 0.026465 0.023471 0.030968 0.048076 0.251265 BC020402 −0.014143 −0.034449 −0.031436 0.004676 0.127490 BC024139 0.037316 −0.009068 0.019397 0.223102 0.008794 Blzf1 0.021608 0.035501 0.048987 0.043345 0.020862 Bola1 −0.000238 −0.023571 −0.011696 0.165591 0.030429 Brd4 0.017478 −0.001701 −0.003086 0.044653 0.090765 Brox −0.012965 −0.006479 −0.024291 0.027580 0.408589 C1galt1c1 −0.037322 −0.035471 −0.025821 0.111284 0.007125 C1ql3 0.037193 0.027143 0.014681 0.872241 0.031389 C330006A16Rik 0.002048 −0.021937 −0.000064 0.107636 0.037658 C77080 −0.012379 −0.018685 −0.022032 0.330408 0.023274 C77370 −0.008607 −0.018310 −0.050701 0.031353 0.879897 Cadm2 −0.031666 0.007890 −0.057798 0.028136 0.492641 Camk1d 0.002103 −0.021799 −0.059723 0.042962 0.630926 Camk2d −0.008343 −0.012511 −0.036507 0.039911 0.438039 Capn3 0.051819 0.028995 0.059326 0.997838 0.020373 Capns2 −0.003440 −0.012476 −0.000049 0.088467 0.033376 Card9 −0.023401 −0.012078 −0.017604 0.546193 0.034352 Carns1 0.024050 0.039077 0.042899 0.081707 0.011544 Cartpt −0.035145 −0.028518 −0.033365 0.073177 0.029756 Casp9 −0.017788 −0.036192 −0.004406 0.539597 0.046217 Casz1 0.002292 0.032986 0.017368 0.020482 0.212097 Cc2d1a 0.097035 0.099738 −0.034594 0.011647 0.502015 Ccbl2 −0.006229 0.000580 0.001696 0.043134 0.054697 Ccdc102a 0.010610 0.025094 0.025529 0.042402 0.398813 Ccdc137 −0.039338 −0.025673 0.003822 0.041148 0.509689 Ccdc162 −0.013561 −0.001696 −0.010546 0.144079 0.027583 Ccdc18 −0.006137 0.022216 0.056094 0.510450 0.026744 Ccdc71l −0.009176 −0.006485 0.005317 0.093585 0.049559 Ccdc88b −0.016783 −0.013084 −0.010888 0.364219 0.042417 Ccne1 0.006161 0.025424 0.051622 0.024593 0.743732 Cd34 −0.026160 −0.009905 0.005988 0.041522 0.203911 Cdc14b −0.001812 −0.022454 −0.006940 0.142560 0.028082 Cdca2 0.104941 0.080389 −0.102077 0.028613 0.652675 Cdh20 0.021184 0.006032 0.069896 0.006466 0.491841 Cdh24 0.042088 0.015338 0.007160 0.030229 0.528470 Cdk6 0.210440 0.004094 0.026616 0.023904 0.917203 Cdr1 0.035166 0.051098 0.010499 0.020388 0.027370 Celsr2 −0.014931 0.014921 −0.028445 0.009650 0.344513 Cenpb 0.015016 0.020412 0.012149 0.021735 0.450704 Cep250 0.018036 0.018775 0.014792 0.048492 0.764264 Cep85l −0.007781 0.011068 −0.013573 0.005658 0.466966 Cerk −0.010458 −0.022563 −0.016745 0.411210 0.019926 Chml −0.004348 0.005401 0.025792 0.423096 0.004320 Cit 0.035513 0.019627 0.016589 0.043276 0.571517 Cklf 0.017535 0.024583 0.025101 0.651082 0.045471 Cks1b −0.029856 −0.029412 −0.022161 0.931788 0.010456 Clic3 0.022312 0.032462 0.057672 0.028433 0.575114 Clic6 0.008745 −0.058586 −0.067127 0.040161 0.867916 Clmn −0.034623 −0.003282 0.017750 0.072765 0.036426 Clock −0.007399 0.001717 0.022307 0.142595 0.048130 Cmc1 −0.002725 0.000092 0.007827 0.037876 0.867438 Cntnap5b 0.028115 0.034920 0.036105 0.049453 0.478204 Coch 0.022825 0.028824 0.025967 0.023138 0.653057 Col7a1 −0.030405 −0.004274 −0.008851 0.603427 0.012553 Commd9 0.017122 0.012919 0.011169 0.193251 0.017034 Cplx2 −0.008452 0.003553 −0.020655 0.181157 0.035215 Cpped1 −0.022119 −0.007738 −0.015496 0.041069 0.287429 Cramp1l 0.011951 0.015843 −0.008129 0.036474 0.128318 Creb3l1 0.026893 0.012759 0.059775 0.002724 0.056047 Creb5 −0.011651 −0.018401 −0.014097 0.214237 0.021926 Crebrf −0.014195 −0.015965 −0.014085 0.026364 0.460324 Cryab 0.002648 0.045479 −0.004698 0.045840 0.373904 Cryzl1 0.022225 0.025653 0.001220 0.012692 0.815966 Ctrl 0.022104 0.018738 0.001256 0.360539 0.047253 Ctsa 0.002983 0.003214 0.014448 0.046033 0.128335 Ctss −0.017689 −0.003842 0.008522 0.093315 0.045646 Cuedc1 −0.000589 0.026350 −0.007672 0.041105 0.117277 Cyb561 −0.006971 −0.019293 −0.021704 0.048446 0.375684 Cyb5a −0.000247 0.012845 0.022998 0.036503 0.249784 Cycs 0.025624 0.038795 0.042975 0.207770 0.005689 Cyp27b1 −0.012760 0.008048 −0.020800 0.099154 0.022989 D030068K23Rik 0.023832 0.056173 0.026594 0.031285 0.793041 D11Wsu47e −0.025550 −0.012995 −0.005751 0.198305 0.015147 Dbx2 −0.014612 −0.011878 −0.008449 0.271487 0.049120 Ddx25 0.011662 0.018683 0.006078 0.016479 0.516485 Ddx27 0.015589 0.003057 0.016676 0.478136 0.003401 Deb1 −0.014179 −0.023216 −0.043748 0.049450 0.947544 Dennd1c 0.039914 0.021151 0.018293 0.526756 0.012549 Dennd2a 0.015498 0.018643 0.017465 0.035084 0.580602 Dimt1 0.023691 −0.003179 0.011145 0.194220 0.033471 Dio3os −0.024838 −0.034409 −0.003964 0.021019 0.463275 Dlx5 0.008132 0.013721 0.015256 0.169939 0.010225 Dmd −0.013903 −0.025774 −0.006702 0.048507 0.714606 Dnaaf2 0.036197 0.008713 0.025264 0.031924 0.673984 Dnah8 −0.016055 0.001490 −0.037444 0.024992 0.264581 Dnase1 0.023842 0.009648 0.013157 0.197763 0.010572 Dok2 0.003324 0.008689 0.025085 0.034923 0.239246 Dpp10 −0.015441 −0.007954 −0.079981 0.046930 0.923224 Dpy19l3 −0.008278 −0.002545 −0.039768 0.035831 0.361002 E130012A19Rik 0.031515 0.049468 0.017424 0.038801 0.968489 E130307A14Rik −0.038934 0.001919 −0.024048 0.583594 0.043410 E2f8 0.026167 0.034633 0.008660 0.009712 0.917045 Ece1 0.015416 0.011281 0.018303 0.017200 0.212793 Efhb −0.017393 −0.024828 −0.031038 0.003463 0.316554 Elk4 0.001924 0.005306 0.002035 0.102711 0.004677 Emp2 0.013323 0.008407 0.019759 0.015086 0.313877 En2 0.010765 0.007161 0.022204 0.028315 0.970905 Epas1 0.008288 0.028333 0.013615 0.046416 0.376189 Ephb2 0.007012 0.015649 0.016260 0.035094 0.363879 Epor 0.027620 −0.013964 −0.026050 0.012732 0.049408 Erbb2 −0.019501 −0.005355 −0.006818 0.105376 0.016819 Esd 0.016940 −0.012689 0.012556 0.073803 0.020485 Exo5 0.003297 0.017595 0.019780 0.275763 0.036255 Exph5 0.001529 0.014966 0.011504 0.022686 0.107954 F11r 0.004515 0.013337 −0.003539 0.025888 0.057087 F8 −0.053619 −0.014388 −0.017645 0.033635 0.718812 Fah −0.001477 −0.013936 −0.017790 0.034176 0.192238 Fam122b 0.014118 0.034620 0.022356 0.680812 0.038356 Fam126a 0.015455 0.001616 0.017247 0.468843 0.048096 Fam160b2 −0.020819 −0.023256 −0.026721 0.359031 0.007140 Fam199x −0.005638 0.010653 0.025052 0.173394 0.048549 Fam19a1 0.021594 0.023319 0.054835 0.004305 0.504384 Fam207a 0.033239 0.023405 −0.002020 0.393933 0.023381 Fam208a 0.014999 0.021287 −0.019797 0.060989 0.011493 Fam20a −0.017786 −0.002093 −0.008460 0.195143 0.036055 Fam43b 0.036932 0.021262 −0.013308 0.012179 0.563795 Fam76b 0.038818 0.043033 0.034589 0.729838 0.010527 Fgf1 −0.018777 −0.018809 −0.024725 0.046123 0.735814 Fkbp1b 0.018456 0.123274 0.042743 0.163818 0.012659 Fndc3c1 −0.025908 −0.035647 −0.017845 0.039584 0.900612 Fndc5 −0.011716 0.002985 −0.030904 0.239289 0.043973 Foxn2 0.015202 0.030201 0.026947 0.481911 0.038159 Frem3 −0.004221 −0.009371 −0.010530 0.136985 0.034447 Fuk 0.017014 0.031916 0.014904 0.046977 0.738661 Fut9 −0.006006 −0.008610 −0.002278 0.035590 0.131621 Gabra2 −0.010751 0.015139 −0.020382 0.024792 0.032847 Gabra4 −0.008958 −0.007002 −0.049786 0.023421 0.316284 Gabrg1 0.034790 0.025006 0.006969 0.056384 0.026992 Gabrg2 0.016472 0.031945 0.004880 0.009039 0.020003 Gale −0.031557 −0.026603 −0.014513 0.717324 0.031829 Galntl6 0.041419 0.018695 0.036613 0.049727 0.932152 Gas2l1 0.022574 0.048417 0.037917 0.011182 0.096764 Gcc1 0.015686 0.013415 0.044273 0.014638 0.069673 Gcnt1 −0.031554 −0.005250 0.017868 0.005124 0.030855 Gemin4 0.012289 0.005809 0.029216 0.036616 0.088258 Glg1 −0.010511 −0.017862 −0.025994 0.021195 0.284436 Gli1 0.047563 0.006019 0.009137 0.027881 0.024868 Glis3 −0.005923 −0.028775 −0.013641 0.333521 0.004438 Glra2 −0.016204 −0.020663 −0.015720 0.351010 0.043765 Gm11346 −0.019898 −0.002056 −0.027717 0.022763 0.920717 Gm11974 −0.014786 −0.027986 −0.018898 0.353110 0.031054 Gm14164 0.019515 0.032459 0.060170 0.045991 0.482338 Gm15880 −0.029035 −0.014696 −0.005096 0.463778 0.026944 Gm16740 0.012690 0.014370 0.067154 0.022138 0.447401 Gm17750 −0.006194 0.027237 0.018766 0.027149 0.330994 Gm17769 0.004824 −0.004512 −0.001662 0.354129 0.047460 Gm20324 −0.005273 0.017530 0.007365 0.023321 0.964918 Gm6981 −0.000080 −0.015038 −0.014025 0.029086 0.677523 Gm7173 0.008156 −0.014436 −0.014937 0.019471 0.976726 Gpatch1 0.431739 −0.003996 0.280313 0.219813 0.041289 Gpm6a −0.002108 −0.004307 −0.014204 0.027061 0.087990 Gpr137 0.017024 0.017506 0.002054 0.048779 0.008619 Gps2 0.028868 0.029826 0.017192 0.033398 0.175325 Grhl3 0.009362 −0.004203 0.020328 0.016927 0.122013 Gria1 −0.013471 −0.008409 −0.069541 0.948533 0.016241 Grik4 0.026827 0.054345 0.052835 0.922786 0.011260 Grin1 0.010083 0.012392 0.080391 0.038090 0.915147 Gsr −0.010725 −0.003364 −0.014168 0.005286 0.015590 Gsta4 0.012697 0.017652 0.043070 0.023978 0.803534 Gstm6 0.002286 0.011797 0.033479 0.580243 0.031027 Gtf3c3 0.090914 0.003015 0.013426 0.011348 0.286818 Gucy1b3 −0.004266 0.009719 −0.006044 0.036949 0.406598 Hcn4 0.019155 0.016333 −0.010748 0.007430 0.013347 Hdgf 0.008440 0.017392 0.027469 0.048896 0.058432 Hebp2 0.008291 0.016743 −0.009318 0.049848 0.160435 Heca 0.001433 −0.001576 −0.006025 0.031399 0.759350 Hist1h2bg −0.019083 −0.027260 −0.008214 0.110010 0.046006 Hist1h4b 0.015243 0.001834 0.041077 0.066468 0.041696 Hist2h3b −0.004677 −0.003625 0.032882 0.020184 0.907305 Hmgb1 0.052492 −0.014904 0.043829 0.035175 0.488319 Hnrnph2 −0.004077 0.003290 0.038536 0.034967 0.116214 Hpca −0.020165 −0.015338 −0.073335 0.770022 0.032902 Hs6st2 −0.028857 −0.013711 −0.032853 0.371762 0.043064 Hsd3b6 0.002284 −0.036960 −0.041971 0.049202 0.961327 Htr2c 0.003401 0.010898 0.046356 0.032362 0.919583 ICa1 −0.001715 −0.012359 −0.047567 0.020965 0.637254 Il1rap 0.025303 0.038021 0.015543 0.004016 0.909725 Inpp5f −0.015269 −0.007679 −0.025605 0.031420 0.393950 Iqcb1 0.016659 0.007740 0.012789 0.007165 0.008668 Irs1 0.011937 0.005462 0.005573 0.037654 0.717298 Irs4 0.011919 0.020235 −0.026328 0.217456 0.035111 Irx2 0.025774 0.000577 0.017257 0.039486 0.253553 Ism1 −0.006999 0.005236 0.002375 0.027306 0.005707 Itga1 −0.010312 0.021954 0.021167 0.046973 0.404084 Jak2 −0.011425 −0.010001 −0.012302 0.049424 0.049844 Kank2 0.028024 0.031366 0.025058 0.164542 0.031171 Kcnd1 0.000315 −0.014348 −0.013143 0.046624 0.385936 Kcnh2 0.018974 0.015479 0.028023 0.036855 0.634312 Kctd5 0.028005 0.009671 0.001124 0.134992 0.026917 Kdm1b −0.008466 −0.004597 0.003408 0.014526 0.123053 Khk 0.027590 0.009110 0.005840 0.816021 0.022254 Kif27 −0.023430 −0.000880 −0.004603 0.024864 0.054733 Kiss1r −0.003762 −0.024762 −0.000403 0.024309 0.304737 Kitl −0.005738 −0.011038 −0.023262 0.157655 0.032926 Kmt2d 0.096876 0.065628 0.005145 0.162448 0.034969 Knstrn −0.020536 0.005269 0.041495 0.037241 0.295019 Kri1 −0.006259 −0.007213 −0.004070 0.008644 0.181103 Lama5 0.011523 0.008852 −0.001478 0.123793 0.025420 Lamtor5 0.001776 0.005946 0.012516 0.015375 0.027874 Lars2 −0.007470 −0.015614 −0.005249 0.027652 0.282781 Lbh −0.011895 −0.007872 −0.009642 0.023526 0.073483 Lingo1 −0.000110 −0.012315 −0.051318 0.108854 0.040087 Lipt2 −0.006800 −0.009957 0.015414 0.014612 0.013521 Lmx1b −0.000908 −0.010401 0.002150 0.188943 0.020214 LOC100862268 −0.016624 −0.012309 −0.017358 0.325547 0.018428 Loxl2 −0.019644 0.063617 0.043249 0.043888 0.047965 Lpcat1 −0.010591 −0.030695 −0.012863 0.046482 0.084511 Lppr4 0.011425 0.009866 0.011001 0.046268 0.472235 Lrfn1 −0.010303 −0.016641 −0.025801 0.668287 0.043557 Lrp1b −0.009740 −0.013339 −0.058090 0.004669 0.342096 Lrp5 0.010065 −0.012394 −0.016613 0.027559 0.016373 Lrr1 −0.006631 −0.005001 −0.007123 0.012557 0.157604 Lrrc29 −0.011883 −0.032442 −0.014291 0.006046 0.586725 Lsm12 −0.016523 −0.014899 −0.023510 0.045080 0.000860 Magel2 −0.004009 −0.017388 −0.010299 0.381163 0.027234 Map1a 0.033386 0.033376 −0.012497 0.746662 0.008371 Map3k14 −0.023962 −0.029427 −0.042339 0.017543 0.746454 Map3k19 −0.000946 0.018588 −0.009143 0.039981 0.515667 Map3k5 −0.080160 −0.077027 −0.080044 0.024002 0.039304 Map7d1 0.018792 0.008053 0.007415 0.783532 0.011803 Mapk8 −0.004944 −0.011569 −0.014343 0.049502 0.018504 Mapkapk2 0.012316 0.007317 0.019279 0.042055 0.348361 Maz 0.014000 0.006374 0.015134 0.017093 0.213478 Mcc 0.023116 0.027314 0.010548 0.020662 0.218258 Mcfd2 0.009132 0.018027 0.003504 0.020501 0.251379 Mdc1 0.038584 −0.004562 0.011400 0.032824 0.393925 Med10 0.008634 −0.009239 0.015578 0.238786 0.049517 Mier2 0.005842 0.008916 0.014886 0.003054 0.188440 Mier3 −0.016152 0.019792 0.016457 0.063984 0.022814 Mir3081 −0.015702 0.000201 −0.014117 0.014512 0.060784 Mir411 −0.020688 −0.001140 −0.009810 0.099324 0.031397 Mir6236 0.012516 −0.003319 0.013316 0.181492 0.034979 Mir6353 0.006485 0.004458 0.005059 0.024869 0.945670 Mllt6 0.035633 0.030711 0.029398 0.015416 0.013526 Mpeg1 −0.008183 0.002809 −0.022620 0.041339 0.100833 Mpnd −0.041822 −0.042997 −0.045820 0.012077 0.148882 Mrpl22 0.020230 −0.003243 0.022213 0.183606 0.048035 Mrps18b 0.016544 0.007835 0.026162 0.016675 0.064731 Mrps33 0.018575 0.010301 0.020973 0.302529 0.044620 Mtcl1 0.024644 0.005838 0.009012 0.662584 0.026472 Mttp −0.012786 −0.016086 −0.000475 0.339442 0.028464 Mtx3 0.000892 0.015037 0.011776 0.047104 0.445012 Mum1 −0.037530 −0.033799 −0.040715 0.048264 0.199749 Mvk −0.024913 −0.025223 −0.010270 0.252348 0.047026 Myh14 0.006294 0.004921 0.021386 0.020653 0.598630 Myo5b −0.021851 −0.007909 −0.015674 0.713086 0.044993 Myo6 −0.030094 −0.019024 −0.032756 0.042033 0.167508 Mzf1 −0.029430 −0.030792 −0.038825 0.314615 0.043194 Nans 0.006464 0.005343 0.015659 0.871903 0.041878 Nap1l5 −0.019423 −0.014022 −0.020209 0.985883 0.013392 Ncmap −0.005604 −0.010358 −0.007121 0.176687 0.035744 Ncor2 −0.029185 −0.031420 −0.051929 0.031998 0.782385 Ndufs4 −0.021712 −0.017029 −0.015262 0.168259 0.046741 Necab1 −0.003495 −0.010480 −0.001512 0.015047 0.041328 Nek11 0.035591 0.003662 −0.003164 0.040074 0.698646 Nexn −0.001135 0.015271 0.041953 0.012171 0.556460 Nfs1 −0.010039 −0.014407 −0.021444 0.286667 0.047529 Nipa2 −0.022149 −0.006146 0.003086 0.368505 0.039745 Nipal2 −0.014866 −0.011007 −0.020556 0.022815 0.280505 Nmd3 −0.002417 −0.014549 −0.011316 0.042559 0.157364 Nol4 −0.011938 −0.007492 −0.016707 0.022873 0.291237 Notch3 0.024110 0.012356 0.058559 0.028682 0.153752 Nptxr 0.009444 −0.001270 −0.037407 0.049716 0.380070 Npy1r −0.005141 −0.031118 −0.040788 0.025919 0.738308 Nr5a2 0.018370 −0.004263 0.063872 0.129014 0.036957 Nrcam −0.027789 −0.036241 0.005793 0.568245 0.040336 Nsun6 −0.024628 −0.024481 −0.007071 0.845330 0.044071 Ntn1 −0.000454 0.004978 −0.022781 0.039911 0.523536 Ntng1 −0.020643 −0.009866 −0.072752 0.018614 0.014514 Nudt11 −0.034167 −0.042052 −0.043198 0.005847 0.110883 Nup214 −0.016064 −0.010920 −0.017894 0.227635 0.015183 Oxsm 0.000108 −0.027689 −0.024744 0.043659 0.656196 Pcdh20 −0.046386 0.011414 −0.051713 0.030321 0.296739 Pcdh7 −0.009136 −0.003067 −0.021611 0.029445 0.358402 Pcdha11 −0.016520 −0.006303 −0.033986 0.717416 0.015041 Pcdha7-g 0.015709 0.021023 0.019379 0.000916 0.055733 Pcdhb12 −0.022938 −0.027371 −0.035569 0.084061 0.046426 Pcdhb20 0.015139 0.023659 −0.028714 0.022486 0.393469 Pcdhb22 −0.026040 0.003917 −0.043745 0.006210 0.731849 Pcdhgb5 −0.006142 −0.031473 −0.043141 0.029102 0.811529 Pde4d −0.016159 −0.022694 −0.035925 0.043676 0.872993 Pdia4 0.014149 0.006092 0.041729 0.042968 0.065241 Pdss2 −0.005670 0.010365 0.003197 0.048344 0.738162 Pex11b −0.011763 −0.031600 −0.021690 0.389610 0.012478 Pgbd1 0.005909 0.027700 0.045936 0.190243 0.037125 Phf5a 0.013185 −0.012260 0.030815 0.015158 0.015097 Phyhip −0.027370 −0.056446 −0.017696 0.096354 0.042792 Pip4k2a −0.005582 0.001053 0.018494 0.155510 0.018271 Pisd-ps3 0.015974 0.014262 0.041224 0.008284 0.084244 Plagl2 −0.024060 −0.038497 −0.053664 0.289714 0.044201 Plcd −0.026596 0.007100 −0.023762 0.372860 0.013936 Pld2 −0.001880 −0.010518 0.005762 0.302955 0.044939 Plek 0.014273 0.035281 0.001704 0.042558 0.134711 Plekhn1 0.001478 0.015303 0.008011 0.003210 0.261697 Plp2 −0.042152 −0.007583 −0.062007 0.026038 0.658918 Plxna3 −0.012183 −0.025374 −0.010284 0.043487 0.564727 Pmaip1 −0.033413 −0.021818 −0.033117 0.002717 0.521253 Poli −0.027254 −0.016261 −0.021448 0.430680 0.033796 Polr3c 0.032982 0.024394 0.034560 0.118620 0.016826 Polr3gl −0.026299 −0.019661 −0.007798 0.015321 0.342353 Polrmt −0.008579 −0.003345 −0.019847 0.088073 0.013188 Pou2f2 0.023079 0.011029 0.023008 0.073404 0.016659 Pou6f2 −0.019664 −0.023395 −0.044026 0.778235 0.038026 Ppap2b −0.013443 −0.018915 −0.041617 0.000165 0.023058 Ppp1r3fos −0.022930 −0.034195 −0.022321 0.177841 0.036070 Prr12 0.000408 0.006244 −0.014256 0.038289 0.218137 Prr36 −0.001649 −0.016707 −0.011756 0.425295 0.020097 Prrc2a 0.028833 0.018199 −0.008305 0.010493 0.492499 Prrg1 −0.029342 −0.025494 −0.039247 0.033721 0.673008 Psg16 −0.034634 −0.027356 −0.043255 0.026303 0.465575 Pstpip2 −0.023110 −0.016239 −0.022393 0.117648 0.014116 Ptch2 0.019629 0.021218 0.017524 0.043590 0.500718 Ptk2b −0.033335 −0.046415 −0.020438 0.024813 0.295167 Ptprf 0.027177 0.012659 0.005862 0.056717 0.017803 Pus7 −0.012930 −0.010325 −0.022733 0.040313 0.162859 Pxn 0.008070 0.027649 0.044857 0.034487 0.376161 Rapgef1 0.026076 0.008201 −0.010935 0.016439 0.161608 Rcan2 −0.006063 −0.002870 −0.042517 0.008899 0.489697 Reps2 −0.002231 0.005908 −0.055310 0.038873 0.146985 Rgs17 −0.018515 −0.027431 −0.040410 0.034399 0.393311 Rhof 0.014475 −0.008579 −0.036521 0.544761 0.031812 Ric8b −0.022708 −0.009367 −0.008797 0.047973 0.057975 Rilpl1 0.018131 0.014790 0.026362 0.029016 0.268459 Rin3 0.018495 0.001943 −0.017445 0.000006 0.000009 Rint1 0.000529 −0.020646 −0.026900 0.178331 0.038604 Rn45s −0.024654 −0.026216 −0.056303 0.023593 0.309697 Rnf126 −0.013695 −0.010554 −0.002962 0.040727 0.066057 Rnpep 0.018020 0.009638 0.014513 0.347253 0.022571 Rnpepl1 0.014489 0.006981 −0.012017 0.792351 0.024831 Rpl19 0.051816 0.002780 −0.007315 0.133847 0.036330 Rpl23a 0.020451 0.039462 0.034281 0.029363 0.197851 Rpl3 0.007858 −0.009074 0.018929 0.422576 0.046796 Rpn2 0.004384 0.007587 0.016861 0.954155 0.011491 Rps12 0.004672 0.011981 0.018951 0.021748 0.521966 Rps2 −0.002592 0.061200 0.044995 0.078156 0.017869 Rwdd2b −0.025276 −0.036682 −0.018135 0.049240 0.612527 Rxfp3 0.003426 −0.016492 −0.010865 0.609581 0.042259 Ryr2 0.006002 −0.014505 −0.067860 0.036844 0.235109 S100a11 0.011627 0.020455 0.031766 0.463287 0.029505 Sall2 0.001304 0.005391 0.019090 0.126702 0.016347 Sarnp 0.024587 0.022710 0.020918 0.015203 0.317385 Sash3 −0.038351 −0.033062 −0.032397 0.048925 0.223167 Scaf1 0.010574 0.029857 0.014365 0.017131 0.535487 Scd4 0.010427 0.007816 0.010159 0.022813 0.340116 Scn1a −0.024593 −0.017361 −0.047242 0.012351 0.015618 Scrib 0.019390 0.013519 0.010277 0.006038 0.169800 Scrt1 0.009206 −0.010709 0.048185 0.018440 0.057750 Scube1 −0.031373 −0.015979 −0.039461 0.402390 0.049442 Scx −0.021443 −0.024279 −0.055883 0.001297 0.018102 Sdha 0.003565 0.013838 0.012220 0.030519 0.813458 Sema3f −0.010080 −0.025308 −0.044520 0.047955 0.410188 Sema6a −0.036419 −0.053792 −0.062240 0.014880 0.000975 Senp7 −0.023767 −0.022385 −0.017452 0.025477 0.982830 Sfxn4 −0.026274 −0.015870 −0.004334 0.030785 0.122576 Sgk3 −0.010629 −0.028230 −0.043582 0.038094 0.818412 Sh3bgrl2 −0.030125 −0.014748 −0.065309 0.043309 0.381214 Sh3bp4 −0.002200 0.015595 0.010299 0.028955 0.614611 Shisa9 0.004435 −0.013156 −0.006440 0.032249 0.231346 Shmt2 0.016896 0.008504 0.033869 0.018573 0.009103 Sigmar1 0.018354 0.032954 0.014076 0.008505 0.283162 Slc10a3 −0.006094 0.037101 0.007231 0.012541 0.622515 Slc15a4 −0.028915 −0.003453 0.003059 0.033385 0.012740 Slc17a5 −0.005964 −0.009700 −0.021747 0.160643 0.002824 Slc1a5 0.006838 −0.018641 0.025391 0.067103 0.023492 Slc22a12 −0.035056 −0.017173 −0.044232 0.033994 0.998381 Slc24a3 −0.029199 −0.031243 −0.034836 0.034300 0.121706 Slc25a1 0.002239 0.000769 −0.018320 0.618569 0.041550 Slc29a4 0.035100 0.035407 0.012864 0.064916 0.031130 Slc35f3 −0.021162 −0.038196 −0.038610 0.210300 0.038326 Slc36a4 −0.032364 −0.036189 −0.038922 0.045337 0.647478 Slc37a2 0.013064 −0.013841 −0.012535 0.024325 0.261275 Slc52a2 0.006082 −0.021065 −0.021000 0.026544 0.305108 Slc6a8 0.013178 0.029358 0.009919 0.034750 0.641018 Slco2b1 0.033816 0.028829 −0.011804 0.046977 0.556896 Slitrk4 −0.026302 −0.011678 −0.009704 0.030259 0.242694 Smarca5-ps −0.046446 −0.010108 −0.014790 0.521651 0.034725 Smim20 −0.008697 −0.033753 −0.019639 0.001939 0.043682 Smim3 0.007351 0.013678 0.019040 0.321499 0.027693 Smim8 −0.004283 0.032803 0.039115 0.410445 0.032207 Smpdl3a −0.023696 −0.021220 −0.050329 0.148628 0.023830 Snhg1 0.001395 0.007754 0.041992 0.040737 0.401207 Snora15 −0.022127 −0.023847 −0.015141 0.226943 0.011575 Snora23 0.000930 0.005307 0.029130 0.027766 0.697849 Snord91a 0.014503 0.027195 0.003526 0.473271 0.042213 Socs6 −0.007457 −0.009155 0.033285 0.736071 0.031995 Sox13 0.022255 0.009403 0.037126 0.105570 0.003372 Sox4 −0.011833 −0.007360 −0.006515 0.039293 0.175793 Spry3 −0.044153 −0.012015 −0.023512 0.465213 0.044173 Spsb1 −0.034845 −0.021166 −0.039115 0.045003 0.271950 Sqrdl −0.016003 −0.003938 −0.005364 0.043374 0.256555 Srbd1 −0.001835 0.018203 0.030946 0.952371 0.012168 Src 0.025614 0.011662 −0.005819 0.071406 0.034419 Srcap 0.013889 0.010037 0.001168 0.033057 0.137941 Srp54b 0.003898 0.033965 0.021679 0.924223 0.030002 St3gal3 0.014004 0.000853 −0.030394 0.009096 0.281437 St8sia6 −0.029909 −0.014567 −0.036468 0.046459 0.906104 Stard5 −0.012664 −0.021311 −0.039125 0.024665 0.079743 Stxbp3-ps −0.041555 −0.034946 −0.010246 0.001381 0.502383 Supt6 0.005264 0.005502 0.001141 0.681687 0.027374 Sv2b −0.027744 0.019267 −0.038717 0.047655 0.525657 Svopl 0.007425 0.033150 0.037057 0.045666 0.443553 Synpo2 0.018899 0.009129 0.038190 0.047874 0.241506 Syt17 −0.016357 −0.011964 −0.018973 0.042350 0.423596 Syt3 −0.003711 0.003187 −0.036472 0.007208 0.195332 Syvn1 0.038976 0.013094 0.000391 0.047307 0.115132 Tacc1 −0.031168 −0.026673 −0.028568 0.826929 0.012318 Tada1 0.007209 −0.009175 −0.013250 0.003895 0.037537 Taf1a 0.001648 0.006096 0.011809 0.022153 0.233880 Taf4a −0.025033 −0.020285 −0.046823 0.005478 0.017439 Taok2 0.013017 0.006513 0.006553 0.015141 0.109765 Tas1r1 −0.030587 −0.031952 −0.033793 0.517938 0.026129 Tatdn1 −0.027544 −0.017623 −0.027386 0.215561 0.009350 Tbc1d10a −0.032363 −0.010165 −0.021231 0.421191 0.032140 Tbc1d4 0.025394 0.060200 0.001359 0.021283 0.067948 Tbc1d9 −0.000923 −0.010408 −0.030709 0.027054 0.039845 Tbx2 −0.009147 0.027857 −0.009265 0.041785 0.641620 Tcf3 0.015857 0.017926 0.036660 0.024555 0.700512 Tenm1 −0.012318 −0.013079 −0.051320 0.008946 0.644578 Tfcp2l1 −0.023127 −0.010180 −0.078130 0.160185 0.021888 Them4 0.016759 0.003096 0.018030 0.131877 0.004031 Thoc7 −0.009868 −0.045513 −0.047251 0.011767 0.084348 Thsd4 −0.027370 −0.025840 −0.055998 0.028647 0.316746 Tigar 0.022699 −0.001033 0.025361 0.883286 0.044757 Timm9 −0.011365 −0.019122 −0.053909 0.005228 0.626517 Tm4sf1 0.008194 0.021260 0.043833 0.387656 0.030942 Tmc7 0.039193 0.002364 0.011948 0.007576 0.141423 Tmem170b 0.029747 0.047088 0.010735 0.024699 0.531595 Tmem180 −0.003963 −0.038750 −0.026556 0.034971 0.013434 Tmem185b −0.017185 −0.036561 −0.043515 0.117839 0.047499 Tmem203 −0.009165 0.019021 0.007152 0.017875 0.111372 Tmem29 −0.024870 0.006120 −0.014121 0.005064 0.035425 Tmem81 −0.011352 −0.013953 0.000315 0.176745 0.048772 Tmem8b 0.012024 −0.012465 −0.026381 0.037704 0.842948 Tmem9 −0.031405 −0.009650 −0.030312 0.077764 0.027167 Tmppe −0.004292 0.003297 −0.016387 0.033296 0.741548 Tnfrsf19 0.013263 0.011495 0.032847 0.010138 0.745693 Tomm5 −0.030245 −0.028115 −0.017621 0.025877 0.534480 Tpcn1 0.030030 0.042158 −0.001416 0.055730 0.035567 Trim13 0.010992 −0.000632 −0.001788 0.019807 0.860509 Trim9 0.016622 0.044166 0.015041 0.022648 0.393678 Tshz3 −0.006809 −0.017449 −0.038020 0.251620 0.020916 Tspan13 0.015132 0.003456 0.011988 0.490731 0.028235 Ttc39aos1 0.012827 0.009621 0.024336 0.013988 0.394096 Tuft1 −0.028904 −0.018103 −0.038787 0.022311 0.801384 Txnrd3 0.017104 −0.003527 −0.020654 0.044973 0.471432 Ubap2 0.010416 0.014737 0.022548 0.131212 0.024075 Ubb 0.027814 −0.001839 −0.001780 0.003985 0.001519 Ube2j2 −0.035874 −0.031261 −0.035263 0.042438 0.731477 Ube2r2 0.014788 0.011837 0.019800 0.016326 0.296872 Uck2 0.003620 0.015707 0.024337 0.177775 0.023297 Utp11l 0.006537 0.021734 0.008165 0.159251 0.046604 Vac14 0.005399 −0.009972 −0.019644 0.338518 0.010671 Vamp7 −0.020817 −0.038301 −0.028122 0.032586 0.196035 Vangl2 0.027570 0.011155 −0.004344 0.014601 0.180576 Vasp 0.022478 0.009889 0.013098 0.060357 0.027992 Vcpip1 −0.000370 0.002315 0.011009 0.064182 0.044061 Vwa8 0.016948 −0.021590 −0.004131 0.010375 0.793385 Vwc2l −0.003988 −0.004835 −0.011561 0.006493 0.055444 Wfs1 −0.017658 0.003797 −0.027011 0.036360 0.126604 Xlr3a −0.001045 0.007115 −0.029520 0.041290 0.002879 Xylt1 −0.001563 −0.034582 −0.036647 0.343656 0.043102 Yipf2 −0.008423 −0.021047 −0.016476 0.011844 0.321436 Zbtb45 −0.009410 −0.032308 −0.026251 0.854544 0.033262 Zbtb46 −0.020206 −0.010232 −0.043049 0.014133 0.275086 Zc3h10 0.022429 0.016654 0.023124 0.011035 0.054442 Zc3h12b −0.017373 −0.022318 −0.048962 0.041581 0.514408 Zc3h18 0.010884 0.005419 0.032800 0.136289 0.006597 Zeb2os −0.016041 0.000340 −0.017544 0.001761 0.130684 Zfhx3 0.007403 0.013639 0.012865 0.551818 0.046705 Zfp212 −0.030576 −0.008674 −0.030651 0.815397 0.038840 Zfp330 −0.038913 −0.038766 −0.026564 0.011652 0.035811 Zfp35 0.005323 0.035270 −0.028103 0.807564 0.016913 Zfp362 0.010752 0.000498 0.009570 0.033042 0.123477 Zfp36l1 −0.004408 0.005355 0.048543 0.039471 0.379769 Zfp628 0.075411 0.012088 0.002199 0.011149 0.177113 Zfp651 −0.000945 −0.008611 −0.019467 0.147411 0.023751 Zfp710 −0.000914 0.022121 0.031224 0.038968 0.540622 Zfp809 −0.027268 −0.025658 −0.034189 0.952685 0.028810 Zfp839 −0.023959 −0.009542 −0.016994 0.466468 0.040120 Zfp85 −0.022880 −0.021387 0.018662 0.042518 0.131547 Zfp850 0.023734 0.022703 0.027762 0.886796 0.028716 Zfpm1 −0.036579 −0.024816 −0.040454 0.003382 0.120900 Zic5 −0.009495 −0.010065 −0.046133 0.832814 0.025530 Zmynd10 −0.034549 −0.026550 −0.023509 0.014819 0.028386 Zscan12 −0.001104 0.005937 0.033403 0.262373 0.036247 Zscan2 0.017638 0.030782 0.035367 0.040146 0.929530 Zswim8 0.015606 0.024358 −0.011696 0.041154 0.222639

Example 6: Table 2 for Examples 1-3, Provided as Parts Tables 2A and 2B

Tables 2A and 2B (collectively “Table 2”) relate 16S rDNA sequencing of SPF vs. Sp fecal microbiota. The “No” in the tables 2A and 2B is used to connect the two tables to each other (e.g., to relate the taxonomic unit of Table 2A to the values in Table 2B, which do not fit into a single table here due to space constraints), and need not correspond to the “No” used in Tables 3A and 3B.

TABLE 2A #OTU ID No Unassigned; Other; Other; Other; Other; Other 1 k_Bacteria; p_Actinobacteria; c_Actinobacteria; o_Bifidobacteriales; 2 f_Bifidobacteriaceae; g_Bifidobacterium k_Bacteria; p_Actinobacteria; c_Coriobacteriia; o_Coriobacteriales; 3 f_Coriobacteriaceae; g k_Bacteria; p_Actinobacteria; c_Coriobacteriia; o_Coriobacteriales; 4 f_Coriobacteriaceae; g_Adlercreutzia k_Bacteria; p_Bacteroidetes; c_Bacteroidia; o_Bacteroidales; f_; g 5 k_Bacteria; p_Bacteroidetes; c_Bacteroidia; o_Bacteroidales; f_Bacteroidaceae; 6 g_Bacteroides k_Bacteria; p_Bacteroidetes; c_Bacteroidia; o_Bacteroidales; 7 f_Porphyromonadaceae; g_Parabacteroides k_Bacteria; p_Bacteroidetes; c_Bacteroidia; o_Bacteroidales; f_Prevotellaceae; 8 g_Prevotella k_Bacteria; p_Bacteroidetes; c_Bacteroidia; o_Bacteroidales; f_Rikenellaceae; 9 g_(—) k_Bacteria; p_Bacteroidetes; c_Bacteroidia; o_Bacteroidales; f_S24-7; g_(—) 10 k_Bacteria; p_Bacteroidetes; c_Bacteroidia; o_Bacteroidales; 11 f_[Odoribacteraceae]; g_Odoribacter k_Bacteria; p_Cyanobacteria; c_4C0d-2; o_MLE1-12; f_; g_(—) 12 k_Bacteria; p_Cyanobacteria; c_4C0d-2; o_YS2; f_; g_(—) 13 k_Bacteria; p_Cyanobacteria; c_Chloroplast; o_Streptophyta; f_; g_(—) 14 k_Bacteria; p_Firmicutes; c_Bacilli; Other; Other; Other 15 k_Bacteria; p_Firmicutes; c_Bacilli; o_Bacillales; Other; Other 16 k_Bacteria; p_Firmicutes; c_Bacilli; o_Bacillales; f_Bacillaceae; g_Bacillus 17 k_Bacteria; p_Firmicutes; c_Bacilli; o_Bacillales; f_Staphylococcaceae; 18 g_Staphylococcus k_Bacteria; p_Firmicutes; c_Bacilli; o_Lactobacillales; f_Enterococcaceae; 19 g_Enterococcus k_Bacteria; p_Firmicutes; c_Bacilli; o_Lactobacillales; f_Lactobacillaceae; 20 g_Lactobacillus k_Bacteria; p_Firmicutes; c_Bacilli; o_Lactobacillales; f_Streptococcaceae; 21 g_Lactococcus k_Bacteria; p_Firmicutes; c_Bacilli; o_Lactobacillales; f_Streptococcaceae; 22 g_Streptococcus k_Bacteria; p_Firmicutes; c_Bacilli; o_Turicibacterales; f_Turicibacteraceae; 23 g_Turicibacter k_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales; Other; Other 24 k_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales; f_; g_(—) 25 k_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales; f_Clostridiaceae; 26 Other k_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales; f_Clostridiaceae; g_(—) 27 k_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales; f_Clostridiaceae; 28 g_Candidatus Arthromitus k_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales; f_Clostridiaceae; 29 g_Clostridium k_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales; f_Dehalobacteriaceae; 30 g_Dehalobacterium k_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales; f_Lachnospiraceae; 31 Other k_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales; f_Lachnospiraceae; g_(—) 32 k_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales; f_Lachnospiraceae; 33 g_Blautia k_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales; f_Lachnospiraceae; 34 g_Coprococcus k_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales; f_Lachnospiraceae; 35 g_Dorea k_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales; f_Lachnospiraceae; 36 g_Roseburia k_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales; f_Lachnospiraceae; 37 g_[Ruminococcus] k_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales; 38 f_Peptostreptococcaceae; g_(—) k_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales; f_Ruminococcaceae; 39 Other k_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales; f_Ruminococcaceae; g_(—) 40 k_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales; f_Ruminococcaceae; 41 g_Oscillospira k_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales; f_Ruminococcaceae; 42 g_Ruminococcus k_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales; f_[Mogibacteriaceae]; 43 g_(—) k_Bacteria; p_Firmicutes; c_Erysipelotrichi; o_Erysipelotrichales; 44 f_Erysipelotrichaceae; g_(—) k_Bacteria; p_Firmicutes; c_Erysipelotrichi; o_Erysipelotrichales; 45 f_Erysipelotrichaceae; g_Allobaculum k_Bacteria; p_Proteobacteria; c_Alphaproteobacteria; o_; f_; g_(—) 46 k_Bacteria; p_Proteobacteria; c_Betaproteobacteria; o_Burkholderiales; 47 f_Alcaligenaceae; g_Sutterella k_Bacteria; p_Proteobacteria; c_Deltaproteobacteria; o_Desulfovibrionales; 48 f_Desulfovibrionaceae; g_(—) k_Bacteria; p_Proteobacteria; c_Deltaproteobacteria; o_Desulfovibrionales; 49 f_Desulfovibrionaceae; g_Bilophila k_Bacteria; p_Proteobacteria; c_Deltaproteobacteria; o_Desulfovibrionales; 50 f_Desulfovibrionaceae; g_Desulfovibrio k_Bacteria; p_Proteobacteria; c_Gammaproteobacteria; o_Enterobacteriales; 51 f_Enterobacteriaceae; Other k_Bacteria; p_Proteobacteria; c_Gammaproteobacteria; o_Enterobacteriales; 52 f_Enterobacteriaceae; g_(—) k_Bacteria; p_Proteobacteria; c_Gammaproteobacteria; o_Enterobacteriales; 53 f_Enterobacteriaceae; g_Morganella k_Bacteria; p_Proteobacteria; c_Gammaproteobacteria; o_Enterobacteriales; 54 f_Enterobacteriaceae; g_Proteus k_Bacteria; p_Proteobacteria; c_Gammaproteobacteria; o_Pseudomonadales; 55 f_Moraxellaceae; g_(—) k_Bacteria; p_Proteobacteria; c_Gammaproteobacteria; o_Pseudomonadales; 56 f_Moraxellaceae; g_Acinetobacter k_Bacteria; p_Tenericutes; c_Mollicutes; o_Anaeroplasmatales; 57 f_Anaeroplasmataceae; g_Anaeroplasma k_Bacteria; p_Tenericutes; c_Mollicutes; o_RF39; f_; g_(—) 58 k_Bacteria; p_Verrucomicrobia; c_Verrucomicrobiae; o_Verrucomicrobiales; 59 f_Verrucomicrobiaceae; g_Akkermansia

TABLE 2B No SPF SPF SPF SPF Sp Sp Sp Sp 1 0.000053 0.000035 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 2 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 3 0.000088 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 4 0.001069 0.001073 0.000369 0.000643 0.000000 0.000000 0.000000 0.000000 5 0.002174 0.000969 0.002277 0.001608 0.000000 0.000000 0.000000 0.000000 6 0.087961 0.035185 0.062068 0.049326 0.000018 0.000014 0.000020 0.000000 7 0.002016 0.002388 0.006137 0.009789 0.000129 0.000086 0.000059 0.000000 8 0.027451 0.008757 0.024723 0.017608 0.000000 0.000000 0.000000 0.000000 9 0.006083 0.065299 0.047169 0.077124 0.000000 0.000043 0.000000 0.000000 10 0.620863 0.237054 0.501377 0.319813 0.000331 0.000229 0.000357 0.000195 11 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 12 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 13 0.001683 0.001160 0.003990 0.000784 0.000000 0.000000 0.000000 0.000000 14 0.000000 0.000000 0.000217 0.000000 0.000000 0.000000 0.000000 0.000000 15 0.000000 0.000000 0.000087 0.000040 0.000000 0.000000 0.000020 0.000000 16 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 17 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 18 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 19 0.000035 0.000017 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 20 0.038231 0.004586 0.025243 0.006854 0.000202 0.000243 0.000258 0.000180 21 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000015 22 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 23 0.001350 0.000485 0.020776 0.008281 0.035306 0.007929 0.000119 0.002869 24 0.000000 0.000000 0.000043 0.000000 0.000644 0.000057 0.000000 0.000015 25 0.085945 0.313621 0.132398 0.249824 0.499917 0.542613 0.509793 0.411759 26 0.000000 0.000000 0.000000 0.000000 0.000754 0.000072 0.000991 0.000075 27 0.001017 0.000588 0.000542 0.000462 0.081099 0.009517 0.039468 0.018443 28 0.024120 0.001021 0.001453 0.000784 0.000000 0.000000 0.000000 0.000000 29 0.000000 0.000000 0.000000 0.000000 0.000975 0.000014 0.000218 0.000165 30 0.000561 0.001315 0.000976 0.002633 0.000846 0.000572 0.000000 0.000000 31 0.000245 0.001004 0.000087 0.000221 0.001490 0.000887 0.013163 0.000315 32 0.038143 0.134285 0.036802 0.103013 0.143194 0.232651 0.139694 0.244785 33 0.000000 0.000017 0.000000 0.000020 0.000000 0.000043 0.000000 0.000030 34 0.001613 0.004431 0.001496 0.001869 0.013339 0.012923 0.016057 0.013517 35 0.000684 0.001713 0.002299 0.005829 0.005041 0.001288 0.003251 0.001877 36 0.000000 0.000017 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 37 0.004645 0.012098 0.006636 0.013447 0.038526 0.026434 0.042124 0.007284 38 0.000018 0.000000 0.000000 0.000020 0.000000 0.000043 0.000000 0.000015 39 0.000070 0.001142 0.000087 0.000382 0.000110 0.001331 0.001388 0.003935 40 0.008116 0.040325 0.019887 0.029768 0.059831 0.073648 0.083733 0.179830 41 0.020281 0.044219 0.015614 0.037024 0.035545 0.063358 0.036990 0.090396 42 0.001630 0.018051 0.008176 0.016080 0.029088 0.017990 0.007929 0.006233 43 0.000000 0.000294 0.000000 0.000181 0.000000 0.000000 0.000000 0.000000 44 0.000824 0.000675 0.000586 0.001990 0.014774 0.002218 0.012449 0.001517 45 0.000088 0.000744 0.003405 0.001508 0.000000 0.000014 0.000000 0.000000 46 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 47 0.009886 0.006352 0.015506 0.013467 0.000000 0.000000 0.000059 0.000000 48 0.000105 0.000952 0.000195 0.001246 0.000000 0.000000 0.000000 0.000000 49 0.000298 0.005175 0.000130 0.001367 0.000000 0.000000 0.000000 0.000000 50 0.001087 0.004050 0.001345 0.011658 0.000000 0.000000 0.000000 0.000000 51 0.000035 0.000017 0.000260 0.000040 0.000055 0.000043 0.000000 0.000030 52 0.000876 0.000121 0.043374 0.008884 0.000129 0.000186 0.000198 0.000240 53 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 54 0.000053 0.000052 0.000065 0.000020 0.000018 0.000072 0.000040 0.000015 55 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 56 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 57 0.008169 0.043008 0.003231 0.000985 0.024212 0.000000 0.034552 0.016235 58 0.002384 0.007667 0.009889 0.005186 0.014406 0.005424 0.057031 0.000015 59 0.000053 0.000035 0.001084 0.000221 0.000018 0.000057 0.000040 0.000015

Example 7: Table 3 for Examples 1-3, Provided as Parts Tables 3A and 3B

Tables 3A and 3B (collectively “Table 3”) relate to fecal 16S rDNA sequencing from BD colonized dams. The “No” in the tables 3A and 3B is used to connect the two tables to each other (e.g., to relate the taxonomic unit of Table 3A to the values in Table 3B, which do not fit into a single table here due to space constraints), and need not correspond to the “No” used in Tables 2A and 2B.

TABLE 3A Taxonomy No Unassigned; Other; Other; Other; Other; Other 1 k_Bacteria; p_Actinobacteria; c_Coriobacteriia; o_Coriobacteriales; 2 f_Coriobacteriaceae; g_Adlercreutzia k_Bacteria; p_Bacteroidetes; c_Bacteroidia; o_Bacteroidales; f_; g_(—) 3 k_Bacteria; p_Bacteroidetes; c_Bacteroidia; o_Bacteroidales; f_Bacteroidaceae; 4 g_Bacteroides k_Bacteria; p_Bacteroidetes; c_Bacteroidia; o_Bacteroidales; 5 f_Porphyromonadaceae; g_Parabacteroides k_Bacteria; p_Bacteroidetes; c_Bacteroidia; o_Bacteroidales; f_Prevotellaceae; 6 g_Prevotella k_Bacteria; p_Bacteroidetes; c_Bacteroidia; o_Bacteroidales; f_Rikenellaceae; 7 g_(—) k_Bacteria; p_Bacteroidetes; c_Bacteroidia; o_Bacteroidales; f_Rikenellaceae; 8 g_AF12 k_Bacteria; p_Bacteroidetes; c_Bacteroidia; o_Bacteroidales; f_S24-7; g_(—) 9 k_Bacteria; p_Bacteroidetes; c_Bacteroidia; o_Bacteroidales; 10 f_[Odoribacteraceae]; g_Odoribacter k_Bacteria; p_Bacteroidetes; c_Bacteroidia; o_Bacteroidales; 11 f_[Paraprevotellaceae]; g_[Prevotella] k_Bacteria; p_Cyanobacteria; c_4C0d-2; o_YS2; f_; g_(—) 12 k_Bacteria; p_Deferribacteres; c_Deferribacteres; o_Deferribacterales; 13 f_Deferribacteraceae; g_Mucispirillum k_Bacteria; p_Firmicutes; c_Bacilli; o_Bacillales; Other; Other 14 k_Bacteria; p_Firmicutes; c_Bacilli; o_Lactobacillales; f_Enterococcaceae; 15 g_Enterococcus k_Bacteria; p_Firmicutes; c_Bacilli; o_Lactobacillales; f_Lactobacillaceae; 16 g_Lactobacillus k_Bacteria; p_Firmicutes; c_Bacilli; o_Turicibacterales; f_Turicibacteraceae; 17 g_Turicibacter k_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales; Other; Other 18 k_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales; f_; g_(—) 19 k_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales; f_Christensenellaceae; 20 g_(—) k_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales; f_Clostridiaceae; 21 Other k_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales; f_Clostridiaceae; g_(—) 22 k_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales; f_Clostridiaceae; 23 g_Candidatus Arthromitus k_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales; f_Clostridiaceae; 24 g_Clostridium k_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales; f_Dehalobacteriaceae; 25 g_Dehalobacterium k_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales; f_Lachnospiraceae; 26 Other k_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales; f_Lachnospiraceae; g_(—) 27 k_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales; f_Lachnospiraceae; 28 g_Blautia k_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales; f_Lachnospiraceae; 29 g_Coprococcus k_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales; f_Lachnospiraceae; 30 g_Dorea k_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales; f_Lachnospiraceae; 31 g_[Ruminococcus] k_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales; f_Peptococcaceae; g_(—) 32 k_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales; 33 f_Peptostreptococcaceae; Other k_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales; 34 f_Peptostreptococcaceae; g_(—) k_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales; f_Ruminococcaceae; g_(—) 35 k_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales; f_Ruminococcaceae; 36 g_Oscillospira k_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales; f_Ruminococcaceae; 37 g_Ruminococcus k_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales; f_[Mogibacteriaceae]; 38 g_(—) k_Bacteria; p_Firmicutes; c_Erysipelotrichi; o_Erysipelotrichales; 39 f_Erysipelotrichaceae; g_(—) k_Bacteria; p_Firmicutes; c_Erysipelotrichi; o_Erysipelotrichales; 40 f_Erysipelotrichaceae; g_Allobaculum k_Bacteria; p_Firmicutes; c_Erysipelotrichi; o_Erysipelotrichales; 41 f_Erysipelotrichaceae; g_Coprobacillus k_Bacteria; p_Firmicutes; c_Erysipelotrichi; o_Erysipelotrichales; 42 f_Erysipelotrichaceae; g_[Eubacterium] k_Bacteria; p_Proteobacteria; c_Alphaproteobacteria; o_; f_; g_(—) 43 k_Bacteria; p_Proteobacteria; c_Alphaproteobacteria; o_RF32; f_; g_(—) 44 k_Bacteria; p_Proteobacteria; c_Betaproteobacteria; o_Burkholderiales; 45 f_Alcaligenaceae; g_Sutterella k_Bacteria; p_Proteobacteria; c_Deltaproteobacteria; o_Desulfovibrionales; 46 f_Desulfovibrionaceae; g_(—) k_Bacteria; p_Proteobacteria; c_Deltaproteobacteria; o_Desulfovibrionales; 47 f_Desulfovibrionaceae; g_Bilophila k_Bacteria; p_Proteobacteria; c_Deltaproteobacteria; o_Desulfovibrionales; 48 f_Desulfovibrionaceae; g_Desulfovibrio k_Bacteria; p_Proteobacteria; c_Epsilonproteobacteria; o_Campylobacterales; 49 f_Helicobacteraceae; Other k_Bacteria; p_Proteobacteria; c_Epsilonproteobacteria; o_Campylobacterales; 50 f_Helicobacteraceae; g_Flexispira k_Bacteria; p_Proteobacteria; c_Epsilonproteobacteria; o_Campylobacterales; 51 f_Helicobacteraceae; g_Helicobacter k_Bacteria; p_Proteobacteria; c_Gammaproteobacteria; o_Enterobacteriales; 52 f_Enterobacteriaceae; g_(—) k_Bacteria; p_Tenericutes; c_Mollicutes; o_Anaeroplasmatales; 53 f_Anaeroplasmataceae; g_Anaeroplasma k_Bacteria; p_Tenericutes; c_Mollicutes; o_RF39; f_; g_(—) 54 k_Bacteria; p_Verrucomicrobia; c_Verrucomicrobiae; o_Verrucomicrobiales; 55 f_Verrucomicrobiaceae; g_Akkermansia

TABLE 3B No BD1 BD2 BD3 BD4 BD5 1 0.000041 0.000115 0.000110 0.000125 0.000145 2 0.000000 0.000000 0.000000 0.000000 0.000000 3 0.000000 0.000000 0.000000 0.000000 0.000000 4 0.913785 0.965081 0.959434 0.961139 0.962127 5 0.000000 0.000000 0.000024 0.000010 0.000026 6 0.000027 0.000012 0.000000 0.000000 0.000000 7 0.000014 0.000023 0.000024 0.000010 0.000043 8 0.000000 0.000000 0.000000 0.000000 0.000000 9 0.000027 0.000058 0.000110 0.000042 0.000120 10 0.000000 0.000000 0.000012 0.000000 0.000000 11 0.000082 0.000023 0.000049 0.000031 0.000009 12 0.000000 0.000000 0.000000 0.000000 0.000000 13 0.000000 0.000012 0.000000 0.000010 0.000009 14 0.000014 0.000058 0.000000 0.001190 0.000026 15 0.000219 0.000230 0.000085 0.000177 0.000060 16 0.000027 0.000035 0.000049 0.000000 0.000009 17 0.000260 0.000219 0.000329 0.000282 0.000273 18 0.001532 0.000622 0.000268 0.000480 0.000316 19 0.000315 0.000219 0.000097 0.000136 0.000111 20 0.000000 0.000000 0.000000 0.000000 0.000000 21 0.001177 0.000426 0.000134 0.000334 0.000333 22 0.000219 0.000092 0.000061 0.000084 0.000077 23 0.000000 0.000000 0.000000 0.000000 0.000000 24 0.000000 0.000000 0.000000 0.000000 0.000000 25 0.000014 0.000000 0.000000 0.000000 0.000000 26 0.000575 0.000703 0.000000 0.000731 0.000265 27 0.000698 0.000346 0.000304 0.000198 0.000171 28 0.016841 0.008710 0.007124 0.005543 0.006968 29 0.005062 0.002431 0.000146 0.002025 0.000837 30 0.000014 0.000000 0.000000 0.000000 0.000000 31 0.000000 0.000012 0.000012 0.000000 0.000009 32 0.000000 0.000000 0.000000 0.000000 0.000000 33 0.000465 0.000184 0.000256 0.000261 0.000239 34 0.046952 0.013779 0.013907 0.016732 0.015354 35 0.000014 0.000012 0.000000 0.000021 0.000009 36 0.000014 0.000012 0.000000 0.000010 0.000017 37 0.000000 0.000000 0.000000 0.000000 0.000000 38 0.000000 0.000000 0.000000 0.000000 0.000000 39 0.000000 0.000012 0.000000 0.000000 0.000000 40 0.000055 0.000012 0.000049 0.000000 0.000026 41 0.000000 0.000000 0.000000 0.000000 0.000000 42 0.002709 0.001682 0.003629 0.001054 0.000666 43 0.000000 0.000000 0.000000 0.000000 0.000000 44 0.000000 0.000000 0.000000 0.000000 0.000000 45 0.000000 0.000000 0.000012 0.000000 0.000000 46 0.000000 0.000012 0.000000 0.000000 0.000017 47 0.000000 0.000000 0.000000 0.000000 0.000000 48 0.000000 0.000000 0.000000 0.000000 0.000000 49 0.000000 0.000000 0.000000 0.000000 0.000000 50 0.000027 0.000000 0.000024 0.000042 0.000009 51 0.000014 0.000000 0.000000 0.000010 0.000017 52 0.008797 0.004862 0.013749 0.009280 0.011716 53 0.000000 0.000000 0.000000 0.000010 0.000000 54 0.000000 0.000000 0.000000 0.000000 0.000000 55 0.000014 0.000012 0.000000 0.000031 0.000000

Example 8: Table 4 for Examples 1-3, Provided as Parts Tables 4A Through 4E

Tables 4A through 4E (collectively “Table 4”) relate to metabolites in E14.5 SPF, ABX, GF, and Sp fetal brains. The cells can be classified from the given data based on p<0.05 or 0.05<p<0.10, as well as based on the mean values being significantly higher or not for each comparison. PSO stands for pathway sort order. Table 4C provides “fold of change,” in which columns 2 through 7 are the ANOVA contrasts, and the “GE” is group effect for one-way ANOVA. The ANOVA contrasts are further provided in Tables 4D and 4E.

TABLE 4A PSO Super Pathway Sub Pathway Biochemical Name 1 Amino_Acid Glycine, Serine and glycine 2 Threonine Metabolism N-acetylglycine 5 dimethylglycine 6 betaine 9 serine 10 N-acetylserine 14 3-phosphoserine 16 threonine 17 N-acetylthreonine 18 allo-threonine 21 homoserine 28 Alanine and Aspartate alanine 30 Metabolism N-acetylalanine 34 aspartate 35 N-acetylaspartate (NAA) 38 asparagine 39 N-acetylasparagine 40 hydroxyasparagine** 41 Glutamate Metabolism glutamate 42 glutamine 43 alpha-ketoglutaramate* 44 N-acetylglutamate 45 N-acetylglutamine 47 4-hydroxyglutamate 50 glutamate, gamma-methyl ester 51 pyroglutamine* 52 N-acetyl-aspartyl-glutamate (NAAG) 53 beta-citrylglutamate 54 gamma-aminobutyrate (GABA) 55 carboxyethyl-GABA 56 N-methyl-GABA 59 S-1-pyrroline-5-carboxylate 65 Histidine Metabolism histidine 66 1-methylhistidine 67 3-methylhistidine 68 N-acetylhistidine 74 imidazole propionate 75 formiminoglutamate 76 imidazole lactate 77 carnosine 80 anserine 82 1-methylhistamine 83 1-methyl-4-imidazoleacetate 84 1-ribosyl-imidazoleacetate* 92 Lysine Metabolism lysine 93 N2-acetyllysine 99 N6-methyllysine 100 N6,N6-dimethyllysine 101 N6,N6,N6-trimethyllysine 102 5-hydroxylysine 103 5-(galactosylhydroxy)-L-lysine 105 2-aminoadipate 107 2-oxoadipate 113 pipecolate 114 6-oxopiperidine-2-carboxylate 117 5-aminovalerate 119 N,N,N-trimethyl-5-aminovalerate 120 Phenylalanine phenylalanine 121 Metabolism N-acetylphenylalanine 122 1-carboxyethylphenylalanine 126 phenyllactate (PLA) 137 Tyrosine Metabolism tyrosine 138 N-acetyltyrosine 150 3-(4-hydroxyphenyl)lactate 154 phenol sulfate 180 o-Tyrosine 181 O-methyltyrosine 191 N-formylphenylalanine 210 Tryptophan Metabolism tryptophan 217 C-glycosyltryptophan 221 kynurenine 227 N-formylanthranilic acid 241 indolelactate 254 3-indoxyl sulfate 261 Leucine, Isoleucine and leucine 263 Valine Metabolism 1-carboxyethylleucine 265 4-methyl-2-oxopentanoate 273 isovalerylcarnitine (C5) 280 beta-hydroxyisovalerate 296 isoleucine 299 1-carboxyethylisoleucine 300 3-methyl-2-oxovalerate 301 alpha-hydroxyisovalerate 303 2-methylbutyrylcarnitine (C5) 306 tiglylcarnitine (C5:1-DC) 311 ethylmalonate 312 methylsuccinate 318 valine 321 1-carboxyethylvaline 323 3-methyl-2-oxobutyrate 326 isobutyrylcarnitine (C4) 328 3-hydroxyisobutyrate 332 Methionine, Cysteine, methionine 333 SAM and Taurine N-acetylmethionine 334 Metabolism N-formylmethionine 337 methionine sulfoxide 342 S-adenosylmethionine (SAM) 343 S-adenosylhomocysteine (SAH) 349 cystathionine 351 cysteine 353 S-methylcysteine 362 cysteine sulfinic acid 363 hypotaurine 364 taurine 365 N-acetyltaurine 367 taurocyamine 369 3-sulfo-L-alanine 371 Urea cycle; Arginine arginine 372 and Proline Metabolism argininosuccinate 373 urea 375 ornithine 379 2-oxoarginine* 380 citrulline 381 homoarginine 382 homocitrulline 383 proline 386 dimethylarginine (SDMA + ADMA) 387 N-acetylarginine 392 N-delta-acetylornithine 397 trans-4-hydroxyproline 399 pro-hydroxy-pro 405 argininate* 410 Creatine Metabolism guanidinoacetate 411 creatine 412 creatinine 418 Polyamine Metabolism putrescine 421 spermidine 426 spermine 429 5-methylthioadenosine (MTA) 430 N-acetylputrescine 436 (N(1) + N(8))-acetylspermidine 438 Guanidino and Acetamido 1-methylguanidine 439 Metabolism 4-guanidinobutanoate 442 Glutathione Metabolism glutathione, reduced (GSH) 443 glutathione, oxidized (GSSG) 445 cysteine-glutathione disulfide 446 S-methylglutathione 447 S-lactoylglutathione 451 5-oxoproline 454 2-hydroxybutyrate/2-hydroxyisobutyrate 456 ophthalmate 459 4-hydroxy-nonenal-glutathione 460 3′-dephospho-CoA-glutathione* 461 CoA-glutathione* 465 Peptide Gamma-glutamyl Amino gamma-glutamylalanine 467 Acid gamma-glutamylglutamate 468 gamma-glutamylglutamine 469 gamma-glutamylglycine 471 gamma-glutamylisoleucine* 472 gamma-glutamylleucine 473 gamma-glutamyl-alpha-lysine 474 gamma-glutamyl-epsilon-lysine 475 gamma-glutamylmethionine 476 gamma-glutamylphenylalanine 477 gamma-glutamylthreonine 478 gamma-glutamyltryptophan 479 gamma-glutamyltyrosine 480 gamma-glutamylvaline 756 Dipeptide prolylglycine 929 Acetylated Peptides phenylacetylglycine 955 Carbohydrate Glycolysis, 1,5-anhydroglucitol (1,5-AG) 958 Gluconeogenesis, and glucose 959 Pyruvate Metabolism glucose 6-phosphate 965 fructose 1,6-diphosphate/ glucose 1,6-diphosphate/ myo-inositol diphosphates 967 dihydroxyacetone phosphate (DHAP) 972 3-phosphoglycerate 973 phosphoenolpyruvate (PEP) 974 pyruvate 975 lactate 978 glycerate 981 Pentose Phosphate 6-phosphogluconate 982 Pathway ribulose 5-phosphate 983 ribose 5-phosphate 984 ribose 1-phosphate 986 sedoheptulose-7-phosphate 993 Pentose Metabolism ribose 994 ribitol 995 ribonate 997 xylulose 5-phosphate 1018 arabitol/xylitol 1020 arabonate/xylonate 1023 sedoheptulose 1024 ribulonate/xylulonate* 1028 Glycogen Metabolism maltotetraose 1030 maltotriose 1033 maltose 1073 Fructose, Mannose and fructose 1078 Galactose Metabolism mannitol/sorbitol 1079 mannose 1080 mannose-6-phosphate 1090 galactose 1-phosphate 1099 galactonate 1104 Nucleotide Sugar UDP-glucose 1108 UDP-galactose 1109 UDP-glucuronate 1111 guanosine 5′-diphospho-fucose 1115 UDP-N-acetylglucosamine/galactosamine 1116 cytidine 5′-monophospho-N- acetylneuraminic acid 1123 Aminosugar Metabolism glucosamine-6-phosphate 1127 glucuronate 1131 N-acetylglucosamine 6-phosphate 1132 N-acetyl-glucosamine 1-phosphate 1138 N-acetylneuraminate 1149 N-acetylglucosaminylasparagine 1150 erythronate* 1152 N-acetylglucosamine/ N-acetylgalactosamine 1157 Energy TCA Cycle citrate 1163 alpha-ketoglutarate 1165 succinylcarnitine (C4-DC) 1166 succinate 1167 fumarate 1169 malate 1179 2-methylcitrate/homocitrate 1181 Oxidative acetylphosphate 1183 Phosphorylation phosphate 1201 Lipid Medium Chain Fatty Acid caproate (6:0) 1203 caprylate (8:0) 1204 pelargonate (9:0) 1205 caprate (10:0) 1217 Long Chain Fatty Acid palmitate (16:0) 1218 palmitoleate (16:1n7) 1223 stearate (18:0) 1225 oleate/vaccenate (18:1) 1236 arachidate (20:0) 1239 eicosenoate (20:1) 1244 erucate (22:1n9) 1247 nervonate (24:1n9)* 1253 Polyunsaturated Fatty hexadecadienoate (16:2n6) 1258 Acid (n3 and n6) eicosapentaenoate (EPA; 20:5n3) 1259 docosapentaenoate (n3 DPA; 22:5n3) 1260 docosahexaenoate (DHA; 22:6n3) 1264 nisinate (24:6n3) 1265 linoleate (18:2n6) 1267 linolenate [alpha or gamma; (18:3n3 or 6)] 1269 dihomo-linolenate (20:3n3 or n6) 1270 arachidonate (20:4n6) 1271 adrenate (22:4n6) 1272 docosapentaenoate (n6 DPA; 22:5n6) 1273 docosadienoate (22:2n6) 1274 dihomo-linoleate (20:2n6) 1276 mead acid (20:3n9) 1277 docosatrienoate (22:3n6)* 1351 Fatty Acid, glutarate (C5-DC) 1355 Dicarboxylate 2-hydroxyglutarate 1359 2-hydroxyadipate 1360 3-hydroxyadipate* 1363 maleate 1372 sebacate (C10-DC) 1430 Fatty Acid, Amino 2-aminoheptanoate 1446 Fatty Acid Metabolism butyrylcarnitine (C4) 1449 (also BCAA Metabolism) propionylcarnitine (C3) 1452 methylmalonate (MMA) 1480 Fatty Acid acetylcarnitine (C2) 1482 Metabolism(Acyl 3-hydroxybutyrylcarnitine (1) 1483 Carnitine) 3-hydroxybutyrylcarnitine (2) 1485 hexanoylcarnitine (C6) 1487 3-hydroxyhexanoylcarnitine (1) 1488 octanoylcarnitine (C8) 1495 laurylcarnitine (C12) 1496 myristoylcarnitine (C14) 1498 palmitoylcarnitine (C16) 1499 palmitoleoylcarnitine (C16:1)* 1500 stearoylcarnitine (C18) 1501 linoleoylcarnitine (C18:2)* 1503 3-hydroxyoleoylcarnitine 1504 oleoylcarnitine (C18:1) 1506 myristoleoylcarnitine (C14:1)* 1517 arachidoylcarnitine (C20)* 1518 arachidonoylcarnitine (C20:4) 1519 adrenoylcarnitine (C22:4)* 1521 meadoylcarnitine (C20:3n9)* 1522 dihomo-linolenoylcarnitine (C20:3n3 or 6)* 1523 dihomo-linoleoylcarnitine (C20:2)* 1524 eicosenoylcarnitine (C20:1)* 1527 docosatrienoylcarnitine (C22:3)* 1528 docosapentaenoylcarnitine (C22:5n3)* 1529 docosahexaenoylcarnitine (C22:6)* 1536 pentadecanoylcarnitine (C15)* 1537 docosapentaenoylcarnitine (C22:5n6)* 1539 3-hydroxypalmitoylcarnitine 1541 Carnitine Metabolism deoxycarnitine 1542 carnitine 1547 Ketone Bodies 3-hydroxybutyrate (BHBA) 1565 Fatty Acid, Monohydroxy 2-hydroxyheptanoate* 1567 2-hydroxydecanoate 1582 3-hydroxydecanoate 1711 Eicosanoid 12-HHTrE 1718 Endocannabinoid oleoyl ethanolamide 1721 palmitoyl ethanolamide 1731 N-stearoyltaurine 1732 N-palmitoyltaurine 1744 palmitoleoyl ethanolamide* 1753 Inositol Metabolism myo-inositol 1754 chiro-inositol 1782 Phospholipid Metabolism choline 1783 choline phosphate 1784 cytidine 5′-diphosphocholine 1786 glycerophosphorylcholine (GPC) 1788 phosphoethanolamine 1789 cytidine-5′-diphosphoethanolamine 1790 glycerophosphoethanolamine 1791 glycerophosphoserine* 1792 glycerophosphoinositol* 1793 trimethylamine N-oxide 1811 Phosphatidylcholine 1-myristoyl-2-palmitoyl-GPC (14:0/16:0) 1815 (PC) 1-myristoyl-2-arachidonoyl-GPC (14:0/20:4)* 1827 1,2-dipalmitoyl-GPC (16:0/16:0) 1828 1-palmitoyl-2-palmitoleoyl-GPC (16:0/16:1)* 1829 1-palmitoyl-2-stearoyl-GPC (16:0/18:0) 1831 1-palmitoyl-2-oleoyl-GPC (16:0/18:1) 1834 1-palmitoyl-2-linoleoyl-GPC (16:0/18:2) 1837 1-palmitoyl-2-gamma-linolenoyl-GPC (16:0/18:3n6)* 1841 1-palmitoyl-2-dihomo-linolenoyl-GPC (16:0/20:3n3 or 6)* 1845 1-palmitoyl-2-arachidonoyl-GPC (16:0/20:4n6) 1851 1-palmitoyl-2-docosahexaenoyl-GPC (16:0/22:6) 1866 1,2-distearoyl-GPC (18:0/18:0) 1867 1-stearoyl-2-oleoyl-GPC (18:0/18:1) 1870 1-stearoyl-2-linoleoyl-GPC (18 :0/18:2)* 1878 1-stearoyl-2-arachidonoyl-GPC (18:0/20:4) 1884 1-stearoyl-2-docosahexaenoyl-GPC (18:0/22:6) 1889 1,2-dioleoyl-GPC (18:1/18:1) 1892 1-oleoyl-2-linoleoyl-GPC (18:1/18:2)* 1902 1-oleoyl-2-docosahexaenoyl-GPC (18:1/22:6)* 1903 1,2-dilinoleoyl-GPC (18:2/18:2) 1907 1-linoleoyl-2-arachidonoyl-GPC (18:2/20:4n6)* 1945 Phosphatidylethanolamine 1,2-dipalmitoyl-GPE (16:0/16:0)* 1948 (PE) 1-palmitoyl-2-stearoyl-GPE (16:0/18:0)* 1949 1-palmitoyl-2-oleoyl-GPE (16:0/18:1) 1950 1-palmitoyl-2-linoleoyl-GPE (16:0/18:2) 1953 1-palmitoyl-2-arachidonoyl-GPE (16:0/20:4)* 1955 1-palmitoyl-2-docosahexaenoyl-GPE (16:0/22:6)* 1962 1-stearoyl-2-oleoyl-GPE (18:0/18:1) 1970 1-stearoyl-2-arachidonoyl-GPE (18:0/20:4) 1974 1-stearoyl-2-docosahexaenoyl-GPE (18:0/22:6)* 1975 1,2-dioleoyl-GPE (18:1/18:1) 1976 1-oleoyl-2-linoleoyl-GPE (18:1/18:2)* 1979 1-oleoyl-2-arachidonoyl-GPE (18:1/20:4)* 1982 1-oleoyl-2-docosahexaenoyl-GPE (18:1/22:6)* 1985 1-linoleoyl-2-arachidonoyl-GPE (18:2/20:4)* 2016 Phosphatidylserine (PS) 1-palmitoyl-2-oleoyl-GPS (16:0/18:1) 2021 1-stearoyl-2-oleoyl-GPS (18:0/18:1) 2024 1-stearoyl-2-arachidonoyl-GPS (18:0/20:4) 2034 Phosphatidylglycerol 1-palmitoyl-2-oleoyl-GPG (16:0/18:1) (PG) 2052 Phosphatidylinositol 1-palmitoyl-2-arachidonoyl-GPI (16:0/20:4)* 2067 (PI) 1-stearoyl-2-arachidonoyl-GPI (18:0/20:4) 2072 1-oleoyl-2-arachidonoyl-GPI (18:1/20:4)* 2105 Lysophospholipid 1-palmitoyl-GPC (16:0) 2106 2-palmitoyl-GPC (16:0)* 2107 1-palmitoleoyl-GPC (16:1)* 2113 1-stearoyl-GPC (18:0) 2115 1-oleoyl-GPC (18:1) 2118 1-linoleoyl-GPC (18:2) 2136 1-arachidonoyl-GPC (20:4n6)* 2157 1-palmitoyl-GPE (16:0) 2164 1-stearoyl-GPE (18:0) 2165 2-stearoyl-GPE (18:0)* 2166 1-oleoyl-GPE (18:1) 2168 1-linoleoyl-GPE (18:2)* 2179 1-arachidonoyl-GPE (20:4n6)* 2192 1-palmitoyl-GPS (16:0)* 2193 1-stearoyl-GPS (18:0)* 2194 1-oleoyl-GPS (18:1) 2199 1-palmitoyl-GPG (16:0)* 2203 1-oleoyl-GPG (18:1)* 2206 1-palmitoyl-GPI (16:0) 2209 1-stearoyl-GPI (18:0) 2211 1-oleoyl-GPI (18:1)* 2217 1-arachidonoyl-GPI (20:4)* 2310 Plasmalogen 1-(1-enyl-palmitoyl)-2-oleoyl-GPE (P-16:0/18:1)* 2311 1-(1-enyl-palmitoyl)-2-linoleoyl-GPE (P-16:0/18:2)* 2312 1-(1-enyl-palmitoyl)-2-palmitoyl-GPC (P-16:0/16:0)* 2313 1-(1-enyl-palmitoyl)-2-palmitoleoyl-GPC (P-16:0/16:1)* 2314 1-(1-enyl-palmitoyl)-2-arachidonoyl-GPE (P-16:0/20:4)* 2318 1-(1-enyl-stearoyl)-2-oleoyl-GPE (P-18:0/18:1) 2327 1-(1-enyl-stearoyl)-2-arachidonoyl-GPE (P-18:0/20:4)* 2343 Lysoplasmalogen 1-(1-enyl-palmitoyl)-GPE (P-16:0)* 2345 1-(1-enyl-oleoyl)-GPE (P-18:1)* 2347 1-(1-enyl-stearoyl)-GPE (P-18:0)* 2348 1-(1-enyl-oleoyl)-2-oleoyl-GPE (P-18:1/18:1)* 2350 Glycerolipid Metabolism glycerol 2351 glycerol 3-phosphate 2356 glycerophosphoglycerol 2357 Monoacylglycerol 1-myristoylglycerol (14:0) 2360 1-palmitoylglycerol (16:0) 2361 1-palmitoleoylglycerol (16:1)* 2364 1-oleoylglycerol (18:1) 2371 1-arachidonylglycerol (20:4) 2374 1-docosahexaenoylglycerol (22:6) 2375 2-myristoylglycerol (14:0) 2376 2-palmitoylglycerol (16:0) 2379 2-oleoylglycerol (18:1) 2381 2-arachidonoylglycerol (20:4) 2383 2-docosahexaenoylglycerol (22:6)* 2394 Diacylglycerol diacylglycerol (14:0/18:1, 16:0/16:1) [2]* 2395 diacylglycerol (16:1/18:2 [2], 16:0/18:3 [1])* 2407 palmitoyl-palmitoyl-glycerol (16:0/16:0) [2]* 2411 palmitoyl-oleoyl-glycerol (16:0/18:1) [2]* 2413 palmitoyl-linoleoyl-glycerol (16:0/18:2) [2]* 2416 palmitoleoyl-oleoyl-glycerol (16:1/18:1) [2]* 2418 palmitoy1-dihomo-linolenoyl-glycerol (16:0/20:3n3 or 6) [2]* 2419 palmitoyl-arachidonoyl-glycerol (16:0/20:4) [1]* 2420 palmitoyl-arachidonoyl-glycerol (16:0/20:4) [2]* 2422 palmitoyl-docosahexaenoyl-glycerol (16:0/22:6) [1]* 2423 palmitoyl-docosahexaenoyl-glycerol (16:0/22:6) [2]* 2426 stearoyl-linoleoyl-glycerol (18:0/18:2) [2]* 2429 oleoyl-oleoyl-glycerol (18:1/18:1) [2]* 2431 oleoyl-linoleoyl-glycerol (18:1/18:2) [1] 2432 oleoyl-linoleoyl-glycerol (18:1/18:2) [2] 2442 stearoyl-arachidonoyl-glycerol (18:0/20:4) [1]* 2443 stearoyl-arachidonoyl-glycerol (18:0/20:4) [2]* 2445 oleoyl-arachidonoyl-glycerol (18:1/20:4) [2]* 2448 stearoyl-docosahexaenoyl-glycerol (18:0/22:6) [1]* 2449 stearoyl-docosahexaenoyl-glycerol (18:0/22:6) [2]* 2474 Sphingolipid Synthesis sphinganine 2480 Dihydroceramides N-palmitoyl-sphinganine (d18:0/16:0) 2481 N-stearoyl-sphinganine (d18:0/18:0)* 2488 Ceramides N-palmitoyl-sphingosine (d18:1/16:0) 2491 N-stearoyl-sphingosine (d18:1/18:0)* 2493 N-arachidoyl-sphingosine (d18:1/20:0)* 2498 N-stearoyl-sphingadienine (d18:2/18:0)* 2499 N-behenoyl-sphingadienine (d18:2/22:0)* 2513 ceramide (d18:1/14:0, d16:1/16:0)* 2514 ceramide (d18:1/17:0, d17:1/18:0)* 2517 ceramide (d16:1/24:1, d18:1/22:1)* 2518 ceramide (d18:2/24:1, d18:1/24:2)* 2520 Hexosylceramides (HCER) glycosyl-N-stearoyl-sphinganine (d18:0/18:0)* 2522 glycosyl-N-palmitoyl-sphingosine (d18:1/16:0) 2523 glycosyl-N-stearoyl-sphingosine (d18:1/18:0) 2525 glycosyl-N-behenoyl-sphingosine (d18:1/22:0)* 2543 glycosyl ceramide (d18:1/20:0, d16:1/22:0)* 2547 glycosyl ceramide (d18:2/24:1, d18:1/24:2)* 2549 Lactosylceramides lactosyl-N-palmitoyl-sphingosine (d18:1/16:0) (LCER) 2561 Dihydrosphingomyelins palmitoyl dihydrosphingomyelin (d18:0/16:0)* 2563 sphingomyelin (d18:0/18:0, d19:0/17:0)* 2564 sphingomyelin (d18:0/20:0, d16:0/22:0)* 2565 Sphingomyelins palmitoyl sphingomyelin (d18:1/16:0) 2566 hydroxypalmitoyl sphingomyelin (d18:1/16:0(OH))** 2567 stearoyl sphingomyelin (d18:1/18:0) 2568 behenoyl sphingomyelin (d18:1/22:0)* 2569 tricosanoyl sphingomyelin (d18:1/23:0)* 2570 lignoceroyl sphingomyelin (d18:1/24:0) 2575 sphingomyelin (d18:1/14:0, d16:1/16:0)* 2577 sphingomyelin (d17:1/16:0, d18:1/15:0, d16:1/17:0)* 2579 sphingomyelin (d18:2/16:0, d18:1/16:1)* 2580 sphingomyelin (d18:1/17:0, d17:1/18:0, d19:1/16:0) 2581 sphingomyelin (d18:1/18:1, d18:2/18:0) 2583 sphingomyelin (d18:1/20:0, d16:1/22:0)* 2588 sphingomyelin (d18:1/22:1, d18:2/22:0, d16:1/24:1)* 2591 sphingomyelin (d18:1/24:1, d18:2/24:0)* 2592 sphingomyelin (d18:2/24:1, d18:1/24:2)* 2594 Sphingosines sphingosine 2610 Mevalonate Metabolism 3-hydroxy-3-methylglutarate 2624 Sterol desmosterol 2625 cholesterol 2649 4-cholesten-3-one 2652 campesterol 2669 7-hydroxycholesterol (alpha or beta) 3453 Nucleotide Purine Metabolism, inosine 5′-monophosphate (IMP) 3454 (Hypo(Xanthine/Inosine inosine 3455 containing hypoxanthine 3456 xanthine 3458 xanthosine 3461 N1-methylinosine 3462 2′-deoxyinosine 3464 urate 3466 allantoin 3471 Purine Metbolism, adenosine 5′-diphosphate (ADP) 3472 Adenine containing adenosine 5′-monophosphate (AMP) 3473 adenosine 3′-monophosphate (3′-AMP) 3474 adenosine 2′-monophosphate (2′-AMP) 3478 adenylosuccinate 3479 adenosine 3480 adenine 3485 N1-methyladenosine 3495 N6-carbamoylthreonyladenosine 3498 2′-deoxyadenosine 5′-monophosphate 3505 N6-succinyladenosine 3507 Purine Metabolism, guanosine 5′-diphosphate (GDP) 3508 Guanine containing guanosine 5′-monophosphate (5′-GMP) 3513 guanosine 3514 guanine 3516 7-methylguanine 3522 N2,N2-dimethylguanosine 3528 2′-deoxyguanosine 5′-monophosphate (dGMP) 3530 2′-deoxyguanosine 3533 N-carbamoylaspartate 3534 Pyrimidine Metabolism, dihydroorotate 3535 Orotate containing orotate 3537 orotidine 3541 Pyrimidine Metabolism, uridine 5′-diphosphate (UDP) 3542 Uracil containing uridine 5′-monophosphate (UMP) 3547 uridine 3548 uracil 3549 pseudouridine 3550 5,6-dihydrouridine 3552 5-methyluridine (ribothymidine) 3563 2′-deoxyuridine 3565 3-ureidoisobutyrate 3566 3-ureidopropionate 3567 beta-alanine 3568 N-acetyl-beta-alanine 3572 Pyrimidine Metabolism, cytidine diphosphate 3573 Cytidine containing cytidine 5′-monophosphate (5′-CMP) 3576 cytidine 2′,3′-cyclic monophosphate 3577 cytidine 3578 cytosine 3579 3-methylcytidine 3580 5-methylcytidine 3585 2′-deoxycytidine 5′-monophosphate 3587 2′-deoxycytidine 3588 2′-O-methylcytidine 3589 5-methyl-2′-deoxycytidine 3598 Pyrimidine Metabolism, thymidine 3599 Thymine containing thymine 3602 3-aminoisobutyrate 3603 Purine and Pyrimidine methylphosphate Metabolism 3607 Dinucleotide (3′-5′)-adenylyluridine 3608 (3′-5′)-adenylyladenosine* 3624 Cofactors and Nicotinate and nicotinamide 3627 Vitamins Nicotinamide Metabolism nicotinamide riboside 3628 nicotinamide adenine dinucleotide (NAD+) 3636 1-methylnicotinamide 3641 trigonelline (N′-methylnicotinate) 3643 N1-Methyl-2-pyridone-5-carboxamide 3644 N1-Methyl-4-pyridone-3-carboxamide 3648 Riboflavin Metabolism riboflavin (Vitamin B2) 3649 flavin adenine dinucleotide (FAD) 3650 flavin mononucleotide (FMN) 3651 Pantothenate and CoA pantothenate 3653 Metabolism phosphopantetheine 3654 3′-dephosphocoenzyme A 3656 coenzyme A 3658 pantetheine 3662 Ascorbate and Aldarate ascorbate (Vitamin C) 3663 Metabolism dehydroascorbate 3664 threonate 3667 oxalate (ethanedioate) 3668 gulonate* 3670 Tocopherol Metabolism alpha-tocopherol 3694 Biotin Metabolism biotin 3698 Folate Metabolism 5-methyltetrahydrofolate (5MeTHF) 3714 Hemoglobin and heme 3715 Porphyrin Metabolism bilirubin (Z,Z) 3718 biliverdin 3727 Thiamine Metabolism thiamin (Vitamin B1) 3728 thiamin monophosphate 3751 Vitamin B6 Metabolism pyridoxamine 3754 pyridoxal 3757 Xenobiotics Benzoate Metabolism hippurate 3771 benzoate 3978 Food Component/Plant 3-formylindole 4000 gluconate 4079 ergothioneine 4108 homostachydrine* 4131 mannonate* 4144 N-glycolylneuraminate 4221 stachydrine 4246 methyl glucopyranoside (alpha + beta) 4274 pyrraline 4363 Bacterial/Fungal tartronate (hydroxymalonate) 4756 Drug - Topical Agents salicylate 4832 Chemical sulfate* 4835 O-sulfo-L-tyrosine 4899 ectoine 4926 phenol red 4930 perfluorooctanesulfonate (PFOS) 4971 thioproline

TABLE 4B PSO Platform Comp ID KEGG HMDB PUBCHEM 1 LC/MS pos early 58 C00037 HMDB00123 750 2 LC/MS pos early 27710 HMDB00532 10972 5 LC/MS pos early 5086 C01026 HMDB00092 673 6 LC/MS pos early 3141 C00719 HMDB00043 247 9 LC/MS pos early 1648 C00065 HMDB00187 5951 10 LC/MS polar 37076 HMDB02931 65249 14 LC/MS pos early 543 C01005 HMDB00272 68841 16 LC/MS pos early 1284 C00188 HMDB00167 6288 17 LC/MS neg 33939 HMDB62557 152204 18 LC/MS polar 15142 C05519 HMDB04041 99289 21 LC/MS polar 18351 C00263 HMDB00719 12647 28 LC/MS pos early 1126 C00041 HMDB00161 5950 30 LC/MS polar 1585 C02847 HMDB00766 88064 34 LC/MS pos early 443 C00049 HMDB00191 5960 35 LC/MS polar 22185 C01042 HMDB00812 65065 38 LC/MS pos early 512 C00152 HMDB00168 6267 39 LC/MS polar 33942 HMDB06028 99715 40 LC/MS pos early 62060 C03124 HMDB32332 97663 41 LC/MS pos early 57 C00025 HMDB00148 611 42 LC/MS pos early 53 C00064 HMDB00641 5961 43 LC/MS polar 62101 44 LC/MS polar 15720 C00624 HMDB01138 70914 45 LC/MS pos early 33943 C02716 HMDB06029 182230 47 LC/MS pos early 40499 C03079 HMDB01344 439902 50 LC/MS pos early 33487 HMDB61715 68662 51 LC/MS pos early 46225 134508 52 LC/MS pos early 35665 C12270 HMDB01067 5255 53 LC/MS neg 54923 C20775 7E+07 54 LC/MS pos early 1416 C00334 HMDB00112 119 55 LC/MS pos early 40007 HMDB02201 2572 56 LC/MS pos early 39577 C15987 70703 59 LC/MS pos early 42370 C04322 HMDB01301 1196 65 LC/MS pos early 59 C00135 HMDB00177 6274 66 LC/MS pos early 30460 C01152 HMDB00001 92105 67 LC/MS pos early 15677 C01152 HMDB00479 64969 68 LC/MS pos early 33946 C02997 HMDB32055 75619 74 LC/MS pos early 40730 HMDB02271 70630 75 LC/MS pos early 43493 C00439 HMDB00854 439233 76 LC/MS pos early 15716 C05568 HMDB02320 440129 77 LC/MS pos early 1768 C00386 HMDB00033 439224 80 LC/MS pos early 15747 C01262 HMDB00194 112072 82 LC/MS pos early 43831 C05127 HMDB00898 3614 83 LC/MS pos early 32350 C05828 HMDB02820 75810 84 LC/MS pos early 61868 C05131 HMDB02331 5E+06 92 LC/MS pos early 1301 C00047 HMDB00182 5962 93 LC/MS polar 36751 C12989 HMDB00446 92907 99 LC/MS pos early 62860 C02728 HMDB02038 164795 100 LC/MS pos early 62862 C05545 HMDB13287 193344 101 LC/MS pos early 1498 C03793 HMDB01325 440120 102 LC/MS pos early 15685 C16741 HMDB00450 1029 103 LC/MS pos early 43582 105 LC/MS neg 6146 C00956 HMDB00510 469 107 LC/MS polar 38347 C00322 HMDB00225 71 113 LC/MS pos early 1444 C00408 HMDB00070 849 114 LC/MS polar 43231 HMDB61705 3E+06 117 LC/MS pos early 18319 C00431 HMDB03355 138 119 LC/MS pos early 57687 120 LC/MS pos early 64 C00079 HMDB00159 6140 121 LC/MS neg 33950 C03519 HMDB00512 74839 122 LC/MS neg 62566 126 LC/MS polar 22130 C05607 HMDB00779 3848 137 LC/MS neg 1299 C00082 HMDB00158 6057 138 LC/MS neg 32390 HMDB00866 68310 150 LC/MS neg 32197 C03672 HMDB00755 9378 154 LC/MS neg 32553 C02180 HMDB60015 74426 180 LC/MS pos early 43392 HMDB06050 91482 181 LC/MS pos early 37451 HMDB14903 76957 191 LC/MS neg 48433 759256 210 LC/MS pos early 54 C00078 HMDB00929 6305 217 LC/MS pos early 48782 1E+07 221 LC/MS pos early 15140 C00328 HMDB00684 161166 227 LC/MS polar 43549 C05653 HMDB04089 101399 241 LC/MS neg 18349 C02043 HMDB00671 92904 254 LC/MS neg 27672 HMDB00682 10258 261 LC/MS pos early 60 C00123 HMDB00687 6106 263 LC/MS neg 62559 265 LC/MS neg 22116 C00233 HMDB00695 70 273 LC/MS pos early 34407 HMDB00688 6E+06 280 LC/MS polar 12129 HMDB00754 69362 296 LC/MS pos early 1125 C00407 HMDB00172 6306 299 LC/MS neg 62558 300 LC/MS neg 15676 C00671 HMDB03736 47 301 LC/MS polar 46537 HMDB00407 99823 303 LC/MS pos early 45095 HMDB00378 6E+06 306 LC/MS pos early 35428 HMDB02366 2E+07 311 LC/MS polar 15765 HMDB00622 11756 312 LC/MS polar 15745 HMDB01844 10349 318 LC/MS pos early 1649 C00183 HMDB00883 6287 321 LC/MS neg 62562 323 LC/MS polar 44526 C00141 HMDB00019 49 326 LC/MS pos early 33441 HMDB00736 168379 328 LC/MS polar 1549 C06001 HMDB00336 87 332 LC/MS pos early 1302 C00073 HMDB00696 6137 333 LC/MS neg 1589 C02712 HMDB11745 448580 334 LC/MS neg 2829 C03145 HMDB01015 439750 337 LC/MS pos early 18374 C02989 HMDB02005 158980 342 LC/MS pos early 15915 C00019 HMDB01185 34756 343 LC/MS neg 42382 C00021 HMDB00939 439155 349 LC/MS pos early 15705 C02291 HMDB00099 439258 351 LC/MS pos early 1868 C00097 HMDB00574 5862 353 LC/MS pos early 39592 HMDB02108 24417 362 LC/MS pos early 37443 C00606 HMDB00996 109 363 LC/MS pos early 590 C00519 HMDB00965 107812 364 LC/MS neg 2125 C00245 HMDB00251 1123 365 LC/MS neg 48187 159864 367 LC/MS neg 35117 C01959 HMDB03584 68340 369 LC/MS polar 47089 C00506 HMDB02757 72886 371 LC/MS pos early 1638 C00062 HMDB00517 232 372 LC/MS pos early 15497 C03406 HMDB00052 828 373 LC/MS pos early 1670 C00086 HMDB00294 1176 375 LC/MS pos early 1493 C00077 HMDB03374 6262 379 LC/MS pos early 55072 C03771 HMDB04225 558 380 LC/MS pos early 2132 C00327 HMDB00904 9750 381 LC/MS pos early 22137 C01924 HMDB00670 9085 382 LC/MS polar 22138 C02427 HMDB00679 65072 383 LC/MS neg 1898 C00148 HMDB00162 145742 386 LC/MS pos early 36808 C03626 HMDB01539 123831 387 LC/MS pos early 33953 C02562 HMDB04620 67427 392 LC/MS neg 43249 1E+07 397 LC/MS pos early 32306 C01157 HMDB00725 5810 399 LC/MS pos early 35127 HMDB06695 1E+07 405 LC/MS pos early 57461 HMDB03148 160437 410 LC/MS pos early 43802 C00581 HMDB00128 763 411 LC/MS pos early 27718 C00300 HMDB00064 586 412 LC/MS pos early 513 C00791 HMDB00562 588 418 LC/MS pos early 1408 C00134 HMDB01414 1045 421 LC/MS pos early 485 C00315 HMDB01257 1102 426 LC/MS pos early 603 C00750 HMDB01256 1103 429 LC/MS pos early 1419 C00170 HMDB01173 439176 430 LC/MS pos early 37496 C02714 HMDB02064 122356 436 LC/MS pos early 57814 438 LC/MS pos early 48114 C02294 HMDB01522 10111 439 LC/MS pos early 15681 C01035 HMDB03464 500 442 LC/MS pos early 2127 C00051 HMDB00125 124886 443 LC/MS pos early 27727 C00127 HMDB03337 65359 445 LC/MS pos early 35159 HMDB00656 4E+06 446 LC/MS pos early 33944 C11347 4E+06 447 LC/MS pos early 15731 C03451 HMDB01066 440018 451 LC/MS neg 1494 C01879 HMDB00267 7405 454 LC/MS polar 52281 456 LC/MS pos early 34592 HMDB05765 7E+06 459 LC/MS neg 48487 460 LC/MS neg 62807 461 LC/MS neg 62804 C00920 1E+07 465 LC/MS pos early 37063 HMDB29142 440103 467 LC/MS pos early 36738 C05282 HMDB11737 92865 468 LC/MS pos early 2730 C05283 HMDB11738 150914 469 LC/MS pos early 33949 HMDB11667 165527 471 LC/MS pos early 34456 HMDB11170 1E+07 472 LC/MS neg 18369 HMDB11171 151023 473 LC/MS pos early 55015 65254 474 LC/MS pos early 33934 HMDB03869 7E+06 475 LC/MS pos early 44872 HMDB29155 7E+06 476 LC/MS neg 33422 HMDB00594 111299 477 LC/MS pos early 33364 HMDB29159 8E+07 478 LC/MS pos early 33947 HMDB29160 4E+06 479 LC/MS pos early 2734 HMDB11741 94340 480 LC/MS pos early 43829 HMDB11172 7E+06 756 LC/MS pos early 40703 HMDB11178 6E+06 929 LC/MS neg 33945 C05598 HMDB00821 68144 955 LC/MS neg 20675 C07326 HMDB02712 64960 958 LC/MS polar 20488 C00031 HMDB00122 79025 959 LC/MS polar 31260 C00668 HMDB01401 5958 965 LC/MS neg 46896 C00354 967 LC/MS neg 15522 C00111 HMDB01473 668 972 LC/MS neg 1414 C00597 HMDB00807 724 973 LC/MS neg 597 C00074 HMDB00263 1005 974 LC/MS polar 22250 C00022 HMDB00243 1060 975 LC/MS polar 527 C00186 HMDB00190 612 978 LC/MS polar 1572 C00258 HMDB00139 752 981 LC/MS neg 15442 C00345 HMDB01316 91493 982 LC/MS polar 1474 C00199 HMDB00618 439184 983 LC/MS polar 561 C00117 HMDB01548 984 LC/MS polar 1763 C00620 HMDB01489 439236 986 LC/MS pos early 35649 C05382 HMDB01068 616 993 LC/MS polar 1471 C00121 HMDB00283 5779 994 LC/MS polar 15772 C00474 HMDB00508 6912 995 LC/MS polar 27731 C01685 HMDB00867 5E+06 997 LC/MS polar 37285 C00231 HMDB00868 439190 1018 LC/MS polar 48885 C01904 6912 1020 LC/MS polar 48255 1023 LC/MS polar 53237 HMDB03219 5E+06 1024 LC/MS polar 61858 1028 LC/MS neg 15910 C02052 HMDB01296 446495 1030 LC/MS neg 44688 C01835 HMDB01262 439586 1033 LC/MS polar 15586 C00208 HMDB00163 1E+07 1073 LC/MS polar 577 C00095 HMDB00660 5984 1078 LC/MS polar 46142 C00794 HMDB00247 5780 1079 LC/MS polar 584 C00159 HMDB00169 18950 1080 LC/MS polar 1469 C00275 HMDB01078 439198 1090 LC/MS polar 15706 C00446 HMDB00645 123912 1099 LC/MS polar 27719 C00880 HMDB00565 128869 1104 LC/MS polar 32344 C00029 HMDB00286 8629 1108 LC/MS polar 15860 C00052 HMDB00302 18068 1109 LC/MS neg 2763 C00167 HMDB00935 17473 1111 LC/MS polar 15903 1115 LC/MS neg 46148 1116 LC/MS polar 36831 C00128 HMDB01176 448209 1123 LC/MS polar 580 C00352 HMDB01254 439217 1127 LC/MS polar 15443 C00191 HMDB00127 444791 1131 LC/MS polar 15107 C00357 HMDB02817 439219 1132 LC/MS polar 15741 C04256 HMDB01367 440364 1138 LC/MS pos early 32377 C00270 HMDB00230 439197 1149 LC/MS pos early 48149 C04540 HMDB00489 123826 1150 LC/MS polar 42420 HMDB00613 3E+06 1152 LC/MS pos early 46539 HMDB00215 24139 1157 LC/MS neg 1564 C00158 HMDB00094 311 1163 LC/MS polar 528 C00026 HMDB00208 51 1165 LC/MS pos early 37058 HMDB61717 7E+07 1166 LC/MS polar 1437 C00042 HMDB00254 1110 1167 LC/MS polar 1643 C00122 HMDB00134 444972 1169 LC/MS neg 1303 C00149 HMDB00156 525 1179 LC/MS neg 52282 1181 LC/MS polar 15488 C00227 HMDB01494 186 1183 LC/MS neg 42109 C00009 HMDB01429 1061 1201 LC/MS neg 32489 C01585 HMDB00535 8892 1203 LC/MS neg 32492 C06423 HMDB00482 379 1204 LC/MS neg 12035 C01601 HMDB00847 8158 1205 LC/MS neg 1642 C01571 HMDB00511 2969 1217 LC/MS neg 1336 C00249 HMDB00220 985 1218 LC/MS neg 33447 C08362 HMDB03229 445638 1223 LC/MS neg 1358 C01530 HMDB00827 5281 1225 LC/MS neg 52285 1236 LC/MS neg 1118 C06425 HMDB02212 10467 1239 LC/MS neg 33587 C16526 HMDB02231 5E+06 1244 LC/MS neg 1552 C08316 HMDB02068 5E+06 1247 LC/MS neg 52674 C08323 HMDB02368 5E+06 1253 LC/MS neg 57652 HMDB00477 1258 LC/MS neg 18467 C06428 HMDB01999 446284 1259 LC/MS neg 32504 C16513 HMDB06528 6E+06 1260 LC/MS neg 44675 C06429 HMDB02183 445580 1264 LC/MS neg 57810 HMDB02007 1E+07 1265 LC/MS neg 1105 C01595 HMDB00673 5E+06 1267 LC/MS neg 34035 C06426 HMDB03073 5E+06 1269 LC/MS neg 35718 C03242 HMDB02925 5E+06 1270 LC/MS neg 1110 C00219 HMDB01043 444899 1271 LC/MS neg 32980 C16527 HMDB02226 5E+06 1272 LC/MS neg 37478 C16513 HMDB01976 6E+06 1273 LC/MS neg 32415 C16533 HMDB61714 5E+06 1274 LC/MS neg 17805 C16525 HMDB05060 6E+06 1276 LC/MS neg 35174 HMDB10378 5E+06 1277 LC/MS neg 57467 1351 LC/MS polar 396 C00489 HMDB00661 743 1355 LC/MS polar 37253 C02630 HMDB00606 43 1359 LC/MS polar 31934 C02360 HMDB00321 193530 1360 LC/MS polar 62069 HMDB00345 151913 1363 LC/MS polar 20676 C01384 HMDB00176 444266 1372 LC/MS polar 32398 C08277 HMDB00792 5192 1430 LC/MS pos early 43761 227939 1446 LC/MS pos early 32412 C02862 HMDB02013 439829 1449 LC/MS pos early 32452 C03017 HMDB00824 107738 1452 LC/MS polar 1496 C02170 HMDB00202 487 1480 LC/MS pos early 32198 C02571 HMDB00201 1 1482 LC/MS pos early 43264 HMDB13127 5E+07 1483 LC/MS pos early 52984 HMDB13127 1485 LC/MS pos late 32328 HMDB00705 6E+06 1487 LC/MS pos early 62557 1488 LC/MS pos late 33936 C02838 HMDB00791 123701 1495 LC/MS pos late 34534 HMDB02250 1E+07 1496 LC/MS pos late 33952 HMDB05066 6E+06 1498 LC/MS pos late 44681 C02990 HMDB00222 461 1499 LC/MS pos late 53223 7E+07 1500 LC/MS pos late 34409 HMDB00848 6E+06 1501 LC/MS pos late 46223 HMDB06469 6E+06 1503 LC/MS pos late 61840 1504 LC/MS pos late 35160 HMDB05065 6E+06 1506 LC/MS pos late 48182 9E+07 1517 LC/MS pos late 57513 HMDB06460 1518 LC/MS pos late 57518 1519 LC/MS pos late 57528 1521 LC/MS pos late 62436 1522 LC/MS pos late 57521 1523 LC/MS pos late 57520 1524 LC/MS pos late 57519 1527 LC/MS pos late 57527 1528 LC/MS pos late 57529 1529 LC/MS pos late 57523 1536 LC/MS pos late 57522 1537 LC/MS pos late 57530 1539 LC/MS pos late 61839 1541 LC/MS pos early 36747 C01181 HMDB01161 134 1542 LC/MS pos early 15500 C00318 HMDB00062 10917 1547 LC/MS polar 542 C01089 HMDB00357 441 1565 LC/MS polar 61827 3E+06 1567 LC/MS neg 42489 21488 1582 LC/MS neg 22053 HMDB02203 26612 1711 LC/MS neg 57393 C20388 HMDB12535 5E+06 1718 LC/MS pos late 38102 HMDB02088 5E+06 1721 LC/MS pos late 38165 C16512 HMDB02100 4671 1731 LC/MS neg 39730 168274 1732 LC/MS neg 39835 1744 LC/MS pos late 57541 HMDB13648 1E+07 1753 LC/MS polar 1124 C00137 HMDB00211 892 1754 LC/MS polar 37112 C19891 HMDB34220 1782 LC/MS pos early 15506 C00114 HMDB00097 305 1783 LC/MS pos early 34396 C00588 HMDB01565 1014 1784 LC/MS polar 34418 C00307 HMDB01413 13804 1786 LC/MS pos early 15990 C00670 HMDB00086 71920 1788 LC/MS pos early 1600 C00346 HMDB00224 1015 1789 LC/MS neg 34410 C00570 HMDB01564 123727 1790 LC/MS pos early 37455 C01233 HMDB00114 123874 1791 LC/MS pos early 57404 3E+06 1792 LC/MS pos early 52307 167572 1793 LC/MS pos early 40406 C01104 HMDB00925 1145 1811 LC/MS pos late 19258 HMDB07869 129657 1815 LC/MS pos late 53195 HMDB07883 1827 LC/MS pos late 19130 HMDB00564 452110 1828 LC/MS pos late 52470 HMDB07969 1829 LC/MS pos late 52616 HMDB07970 1831 LC/MS pos late 52461 HMDB07972 6E+06 1834 LC/MS pos late 42446 HMDB07973 5E+06 1837 LC/MS pos late 54812 HMDB07974 1841 LC/MS pos late 52454 1845 LC/MS pos late 52462 HMDB07982 1E+07 1851 LC/MS pos late 52610 HMDB07991 6E+06 1866 LC/MS pos late 19132 HMDB08036 94190 1867 LC/MS pos late 52438 HMDB08038 1870 LC/MS pos late 52452 HMDB08039 1878 LC/MS pos late 42450 HMDB08048 2E+07 1884 LC/MS pos late 52611 HMDB08057 1889 LC/MS pos late 52457 1E+07 1892 LC/MS pos late 52453 1902 LC/MS pos late 52697 HMDB08123 1903 LC/MS pos late 52603 HMDB08138 5E+06 1907 LC/MS pos late 52710 HMDB08147 1945 LC/MS pos late 57341 HMDB08923 445468 1948 LC/MS pos late 57388 HMDB08925 5E+06 1949 LC/MS pos late 19263 HMDB05320 5E+06 1950 LC/MS pos late 42449 HMDB05322 1E+07 1953 LC/MS pos late 52464 HMDB05323 1E+07 1955 LC/MS pos late 52465 HMDB05324 1E+07 1962 LC/MS pos late 42448 HMDB08993 1970 LC/MS pos late 52447 HMDB09003 5E+06 1974 LC/MS pos late 52466 HMDB05334 1E+07 1975 LC/MS pos late 52609 1E+07 1976 LC/MS pos late 52687 HMDB05349 1E+07 1979 LC/MS pos late 55041 HMDB09069 1982 LC/MS pos late 53209 1985 LC/MS pos late 53189 HMDB09102 2016 LC/MS pos late 19261 C13880 HMDB12357 5E+06 2021 LC/MS pos late 19265 HMDB10163 1E+07 2024 LC/MS pos late 52235 HMDB12383 2034 LC/MS pos late 52448 5E+06 2052 LC/MS pos late 52467 HMDB09789 2067 LC/MS pos late 52449 HMDB09815 2072 LC/MS pos late 54994 HMDB09844 2105 LC/MS pos late 33955 HMDB10382 86554 2106 LC/MS pos late 35253 HMDB61702 2E+07 2107 LC/MS pos late 33230 HMDB10383 2E+07 2113 LC/MS pos late 33961 HMDB10384 497299 2115 LC/MS pos late 48258 HMDB02815 2E+07 2118 LC/MS pos late 34419 C04100 HMDB10386 1E+07 2136 LC/MS neg 34061 C05208 HMDB10395 2157 LC/MS pos late 35631 HMDB11503 1E+07 2164 LC/MS pos late 42398 HMDB11130 1E+07 2165 LC/MS neg 41220 HMDB11129 2166 LC/MS pos late 35628 HMDB11506 1E+07 2168 LC/MS pos late 36600 HMDB11507 5E+07 2179 LC/MS neg 35186 HMDB11517 4E+07 2192 LC/MS neg 46130 1E+07 2193 LC/MS neg 45966 1E+07 2194 LC/MS neg 19260 HMDB61694 1E+07 2199 LC/MS neg 45970 3E+06 2203 LC/MS neg 45968 2206 LC/MS neg 35305 HMDB61695 2209 LC/MS neg 19324 HMDB61696 2211 LC/MS neg 36602 2217 LC/MS neg 34214 HMDB61690 2310 LC/MS pos late 52477 HMDB11342 2311 LC/MS pos late 52677 HMDB11343 2312 LC/MS pos late 52716 HMDB11206 1E+07 2313 LC/MS pos late 52713 HMDB11207 2314 LC/MS pos late 52673 HMDB11352 2318 LC/MS pos late 52614 HMDB11375 2327 LC/MS pos late 52475 HMDB05779 1E+07 2343 LC/MS pos late 39270 2345 LC/MS pos late 44621 2347 LC/MS pos late 39271 2348 LC/MS pos late 54691 HMDB11441 2350 LC/MS neg 15122 C00116 HMDB00131 753 2351 LC/MS pos early 43847 C00093 HMDB00126 754 2356 LC/MS polar 48857 C03274 439964 2357 LC/MS neg 35625 C01885 HMDB11561 79050 2360 LC/MS neg 21127 HMDB31074 14900 2361 LC/MS neg 52431 HMDB11565 2364 LC/MS neg 21184 HMDB11567 5E+06 2371 LC/MS neg 34397 C13857 HMDB11549 5E+06 2374 LC/MS neg 35153 HMDB11587 2375 LC/MS neg 34383 HMDB11530 137938 2376 LC/MS neg 33419 HMDB11533 123409 2379 LC/MS neg 21232 HMDB11537 5E+06 2381 LC/MS neg 19266 C13856 HMDB04666 5E+06 2383 LC/MS neg 48675 HMDB11557 2394 LC/MS pos late 54954 2395 LC/MS pos late 54966 2407 LC/MS pos late 54990 HMDB07098 2411 LC/MS pos late 54942 C13861 HMDB07102 2413 LC/MS pos late 52634 HMDB07103 2416 LC/MS pos late 52631 2418 LC/MS pos late 54941 2419 LC/MS pos late 54957 HMDB07112 2420 LC/MS pos late 54958 HMDB07112 2422 LC/MS pos late 57373 HMDB07121 2423 LC/MS pos late 57374 HMDB07121 2426 LC/MS pos late 54947 2429 LC/MS pos late 54946 HMDB07218 2431 LC/MS pos late 46798 HMDB07219 2432 LC/MS pos late 46799 HMDB07219 2442 LC/MS pos late 57450 2443 LC/MS pos late 57449 2445 LC/MS pos late 54961 HMDB07228 2448 LC/MS pos late 57387 2449 LC/MS pos late 57368 2474 LC/MS pos late 17769 C00836 HMDB00269 3126 2480 LC/MS pos late 52604 HMDB11760 5E+06 2481 LC/MS pos late 1759 5E+06 2488 LC/MS pos late 44877 HMDB04949 5E+06 2491 LC/MS pos late 54979 HMDB04950 5E+06 2493 LC/MS pos late 57424 HMDB04951 5E+06 2498 LC/MS pos late 57417 2499 LC/MS pos late 57372 2513 LC/MS pos late 57432 2514 LC/MS pos late 57434 2517 LC/MS pos late 57437 2518 LC/MS pos late 57443 2520 LC/MS pos late 57418 6E+06 2522 LC/MS pos late 53013 2523 LC/MS pos late 52234 2525 LC/MS pos late 57371 2543 LC/MS pos late 57595 2547 LC/MS pos late 57453 2549 LC/MS pos late 53010 2561 LC/MS pos late 52434 1E+07 2563 LC/MS pos late 57473 HMDB12087 2564 LC/MS pos late 57476 2565 LC/MS pos late 37506 1E+07 2566 LC/MS pos late 62851 2567 LC/MS pos late 19503 C00550 HMDB01348 6E+06 2568 LC/MS pos late 48492 HMDB12103 2569 LC/MS pos late 52436 HMDB12105 2570 LC/MS pos late 57330 2575 LC/MS pos late 42463 HMDB12097 1E+07 2577 LC/MS pos late 52433 2579 LC/MS pos late 42459 2580 LC/MS pos late 52615 2581 LC/MS pos late 37529 HMDB12101 6E+06 2583 LC/MS pos late 48490 HMDB12102 2588 LC/MS pos late 48493 HMDB12104 2591 LC/MS pos late 47153 HMDB12107 2592 LC/MS pos late 52437 2594 LC/MS pos late 17747 C00319 HMDB00252 5E+06 2610 LC/MS polar 531 C03761 HMDB00355 1662 2624 LC/MS pos late 6065 C01802 HMDB02719 439577 2625 LC/MS pos late 63 C00187 HMDB00067 1E+07 2649 LC/MS pos late 38125 C00599 HMDB00921 91477 2652 LC/MS pos late 33997 C01789 HMDB02869 173183 2669 LC/MS pos late 47890 HMDB06119 107722 3453 LC/MS pos early 2133 C00130 HMDB00175 8582 3454 LC/MS neg 1123 C00294 HMDB00195 6021 3455 LC/MS pos early 3127 C00262 HMDB00157 790 3456 LC/MS pos early 3147 C00385 HMDB00292 1188 3458 LC/MS neg 15136 C01762 HMDB00299 64959 3461 LC/MS pos early 48351 HMDB02721 65095 3462 LC/MS neg 15076 C05512 HMDB00071 65058 3464 LC/MS neg 1604 C00366 HMDB00289 1175 3466 LC/MS pos early 1107 C02350 HMDB00462 204 3471 LC/MS neg 3108 C00008 HMDB01341 6022 3472 LC/MS pos early 32342 C00020 HMDB00045 6083 3473 LC/MS neg 35142 C01367 HMDB03540 41211 3474 LC/MS neg 36815 C00946 HMDB11617 94136 3478 LC/MS neg 57838 C03794 HMDB00536 195 3479 LC/MS pos early 555 C00212 HMDB00050 60961 3480 LC/MS pos early 554 C00147 HMDB00034 190 3485 LC/MS pos early 15650 C02494 HMDB03331 27476 3495 LC/MS neg 35157 HMDB41623 161466 3498 LC/MS neg 46333 C00360 HMDB00905 12599 3505 LC/MS pos early 48130 HMDB00912 165243 3507 LC/MS neg 2848 C00035 HMDB01201 8977 3508 LC/MS pos early 2849 C00144 HMDB01397 6804 3513 LC/MS pos early 1573 C00387 HMDB00133 6802 3514 LC/MS pos early 32352 C00242 HMDB00132 764 3516 LC/MS pos early 35114 C02242 HMDB00897 11361 3522 LC/MS pos early 35137 HMDB04824 92919 3528 LC/MS pos early 35662 C00362 HMDB01044 65059 3530 LC/MS neg 1411 C00330 HMDB00085 187790 3533 LC/MS polar 1594 C00438 HMDB00828 93072 3534 LC/MS polar 601 C00337 HMDB03349 648 3535 LC/MS polar 1505 C00295 HMDB00226 967 3537 LC/MS polar 35172 HMDB00788 92751 3541 LC/MS neg 5345 C00015 HMDB00295 6031 3542 LC/MS pos early 2856 C00105 HMDB00288 6030 3547 LC/MS neg 606 C00299 HMDB00296 6029 3548 LC/MS neg 605 C00106 HMDB00300 1174 3549 LC/MS neg 33442 C02067 HMDB00767 15047 3550 LC/MS neg 61833 94312 3552 LC/MS neg 35136 HMDB00884 445408 3563 LC/MS neg 52602 C00526 HMDB00012 13712 3565 LC/MS pos early 57549 C05100 HMDB02031 160663 3566 LC/MS pos early 3155 C02642 HMDB00026 111 3567 LC/MS pos early 55 C00099 HMDB00056 239 3568 LC/MS polar 37432 C01073 76406 3572 LC/MS neg 2841 C00112 HMDB01546 6132 3573 LC/MS pos early 2372 C00055 HMDB00095 6131 3576 LC/MS neg 37465 C02354 HMDB11691 417654 3577 LC/MS pos early 514 C00475 HMDB00089 6175 3578 LC/MS pos early 573 C00380 HMDB00630 597 3579 LC/MS pos early 35132 159649 3580 LC/MS pos early 22119 HMDB00982 92918 3585 LC/MS pos early 533 C00239 HMDB01202 13945 3587 LC/MS pos early 15949 C00881 HMDB00014 13711 3588 LC/MS pos early 57554 150971 3589 LC/MS pos early 38159 C03592 HMDB02224 440055 3598 LC/MS neg 2183 C00214 HMDB00273 5789 3599 LC/MS neg 604 C00178 HMDB00262 1135 3602 LC/MS pos early 1566 C05145 HMDB03911 64956 3603 LC/MS pos early 37070 HMDB61711 13130 3607 LC/MS neg 52740 112074 3608 LC/MS neg 62396 94231 3624 LC/MS pos early 594 C00153 HMDB01406 936 3627 LC/MS pos early 33013 C03150 HMDB00855 439924 3628 LC/MS neg 5278 C00003 HMDB00902 5893 3636 LC/MS pos early 27665 C02918 HMDB00699 1E+07 3641 LC/MS pos early 32401 C01004 HMDB00875 5570 3643 LC/MS neg 40469 C05842 HMDB04193 69698 3644 LC/MS pos early 57584 C05843 HMDB04194 440810 3648 LC/MS neg 1827 C00255 HMDB00244 493570 3649 LC/MS neg 2134 C00016 HMDB01248 643975 3650 LC/MS neg 15797 C00061 HMDB01520 710 3651 LC/MS pos early 1508 C00864 HMDB00210 6613 3653 LC/MS neg 15504 C01134 HMDB01416 987 3654 LC/MS neg 18289 C00882 HMDB01373 444485 3656 LC/MS neg 46322 C00010 HMDB01423 317 3658 LC/MS polar 57555 C00831 439322 3662 LC/MS pos early 32354 C00072 HMDB00044 3663 LC/MS polar 1659 C05422 HMDB01264 835 3664 LC/MS polar 27738 C01620 HMDB00943 151152 3667 LC/MS neg 20694 C00209 HMDB02329 971 3668 LC/MS polar 46957 C00257 HMDB03290 1E+07 3670 LC/MS pos late 1561 C02477 HMDB01893 14985 3694 LC/MS pos early 568 C00120 HMDB00030 171548 3698 LC/MS neg 18330 C00440 HMDB01396 146 3714 LC/MS pos late 41754 C00032 HMDB03178 26945 3715 LC/MS pos late 43807 C00486 HMDB00054 5E+06 3718 LC/MS pos late 2137 C00500 HMDB01008 5E+06 3727 LC/MS pos early 5341 C00378 HMDB00235 1130 3728 LC/MS neg 15798 C01081 HMDB02666 3E+06 3751 LC/MS pos early 2150 C00534 HMDB01431 1052 3754 LC/MS pos early 1651 C00250 HMDB01545 1050 3757 LC/MS neg 15753 C01586 HMDB00714 464 3771 LC/MS neg 15778 C00180 HMDB01870 243 3978 LC/MS pos early 62863 C08493 HMDB29737 10256 4000 LC/MS polar 587 C00257 HMDB00625 10690 4079 LC/MS pos early 37459 C05570 HMDB03045 3E+06 4108 LC/MS pos early 33009 C08283 HMDB33433 441447 4131 LC/MS polar 62864 3E+06 4144 LC/MS pos early 37123 C03410 HMDB00833 123802 4221 LC/MS pos early 34384 C10172 HMDB04827 115244 4246 LC/MS neg 46144 4274 LC/MS pos early 48428 HMDB33143 122228 4363 LC/MS neg 20693 C02287 HMDB35227 45 4756 LC/MS polar 1515 C00805 HMDB01895 338 4832 LC/MS neg 46960 C00059 HMDB01448 1118 4835 LC/MS neg 45413 514186 4899 LC/MS pos early 35651 C06231 126041 4926 LC/MS neg 36817 C12600 4766 4930 LC/MS polar 57564 C18142 HMDB59586 74483 4971 LC/MS pos early 53231 93176

TABLE 4C Sp/ ABX/ GF/ Sp/ GF/ GF/ PSO SPF SPF SPF ABX ABX Sp GE 1 0.83 0.82 0.94 1.01 1.15 1.14 2 0.91 1.01 1.38 0.91 1.37 1.51 5 0.76 0.69 0.83 1.10 1.20 1.09 Y 6 0.81 0.66 0.80 1.23 1.21 0.98 Y 9 0.85 1.07 1.04 0.79 0.97 1.23 10 0.80 0.82 0.90 0.98 1.10 1.12 Y 14 0.80 1.01 1.53 0.79 1.51 1.91 16 0.79 0.80 0.92 0.98 1.14 1.17 17 0.90 0.84 1.01 1.07 1.19 1.12 18 0.79 0.68 0.82 1.16 1.21 1.04 21 0.39 0.29 0.52 1.32 1.78 1.35 Y 28 0.82 0.83 0.86 0.99 1.04 1.05 y 30 0.82 0.72 0.83 1.15 1.16 1.01 Y 34 0.82 0.81 0.83 1.01 1.03 1.01 Y 35 0.79 0.69 0.73 1.15 1.06 0.92 Y 38 0.95 0.95 0.96 1.00 1.01 1.01 39 0.92 0.87 0.98 1.06 1.13 1.07 40 0.69 0.55 0.56 1.27 1.03 0.81 Y 41 0.87 0.84 0.86 1.04 1.03 0.99 Y 42 0.86 0.70 0.67 1.23 0.96 0.78 Y 43 1.87 0.77 0.63 2.42 0.81 0.34 Y 44 0.77 0.76 0.75 1.02 1.00 0.98 y 45 0.83 0.70 0.69 1.18 0.97 0.83 Y 47 0.64 1.04 1.05 0.61 1.01 1.65 Y 50 0.82 0.71 0.84 1.16 1.19 1.02 51 0.97 0.73 0.51 1.34 0.70 0.52 Y 52 0.84 0.80 0.74 1.05 0.93 0.88 Y 53 0.88 0.85 0.81 1.04 0.95 0.92 Y 54 0.80 0.75 0.72 1.06 0.96 0.90 Y 55 0.83 0.88 0.60 0.94 0.68 0.72 56 0.76 1.22 0.72 0.62 0.59 0.95 59 0.81 1.70 0.63 0.47 0.37 0.78 y 65 0.96 1.14 0.96 0.85 0.85 1.00 66 0.81 1.35 0.81 0.60 0.60 1.00 Y 67 0.73 1.01 0.70 0.72 0.70 0.96 68 0.70 0.94 1.02 0.74 1.08 1.46 y 74 1.60 0.33 0.33 4.81 0.99 0.20 Y 75 1.22 1.80 1.53 0.68 0.85 1.26 y 76 0.90 0.81 0.76 1.11 0.94 0.84 Y 77 0.39 0.34 0.52 1.15 1.53 1.33 Y 80 0.82 0.40 0.42 2.04 1.04 0.51 Y 82 0.42 0.27 0.22 1.54 0.82 0.53 y 83 0.51 0.36 0.41 1.42 1.15 0.81 y 84 0.33 0.43 0.36 0.76 0.85 1.11 Y 92 0.89 0.90 0.94 0.98 1.04 1.06 93 0.88 0.49 0.58 1.80 1.18 0.66 Y 99 0.86 0.64 0.57 1.35 0.89 0.66 Y 100 0.75 0.57 0.57 1.30 0.99 0.76 Y 101 0.73 0.61 0.65 1.19 1.06 0.90 Y 102 0.66 0.79 0.66 0.83 0.83 1.00 Y 103 0.82 0.77 0.82 1.07 1.06 0.99 Y 105 0.85 1.05 1.07 0.80 1.02 1.27 107 1.01 1.09 1.50 0.93 1.38 1.49 113 0.75 0.57 0.47 1.32 0.83 0.63 Y 114 0.96 1.02 1.70 0.94 1.66 1.77 117 0.98 0.85 0.60 1.16 0.71 0.62 119 1.79 0.09 0.16 19.45 1.71 0.09 Y 120 0.90 0.82 0.79 1.10 0.96 0.88 Y 121 1.09 0.83 0.81 1.31 0.98 0.74 122 0.95 0.79 0.82 1.19 1.03 0.87 126 0.73 0.60 0.58 1.22 0.96 0.79 y 137 0.97 0.89 0.79 1.10 0.90 0.82 y 138 0.85 0.75 0.52 1.14 0.70 0.61 Y 150 1.01 0.81 0.79 1.24 0.97 0.78 Y 154 0.63 0.44 0.44 1.43 1.00 0.70 Y 180 0.91 0.95 1.00 0.96 1.04 1.09 181 0.88 0.91 1.07 0.97 1.19 1.22 191 0.98 0.40 0.77 2.41 1.90 0.79 210 0.79 0.81 0.77 0.98 0.95 0.97 Y 217 0.78 0.71 0.73 1.11 1.03 0.93 Y 221 0.91 0.86 0.82 1.06 0.95 0.89 227 0.85 0.34 0.88 2.49 2.60 1.04 241 0.74 0.48 0.59 1.56 1.24 0.80 254 0.56 0.31 0.08 1.79 0.25 0.14 Y 261 0.86 0.82 0.81 1.04 0.98 0.95 y 263 0.92 0.78 0.97 1.18 1.24 1.05 265 1.33 1.28 1.26 1.04 0.98 0.95 273 0.95 0.90 1.12 1.05 1.25 1.18 280 0.72 0.89 0.80 0.81 0.90 1.11 Y 296 0.87 0.77 0.77 1.13 1.01 0.89 Y 299 1.15 0.75 0.66 1.54 0.89 0.58 300 1.35 0.96 1.21 1.40 1.26 0.90 301 0.64 0.83 0.71 0.77 0.85 1.10 Y 303 0.95 0.90 0.97 1.05 1.07 1.02 306 0.87 0.83 0.87 1.04 1.05 1.01 311 0.89 0.69 0.81 1.30 1.18 0.90 Y 312 0.72 0.67 0.77 1.08 1.15 1.06 Y 318 0.87 0.85 0.91 1.02 1.07 1.05 321 0.95 0.69 0.88 1.37 1.27 0.92 y 323 1.23 1.13 1.21 1.09 1.07 0.98 326 1.01 1.03 1.20 0.99 1.17 1.18 328 0.80 0.76 1.02 1.06 1.34 1.27 332 0.74 0.68 0.69 1.09 1.02 0.94 Y 333 1.03 0.69 0.89 1.49 1.28 0.86 Y 334 0.82 0.86 0.85 0.95 0.98 1.03 y 337 0.77 0.47 0.73 1.65 1.57 0.95 Y 342 0.87 0.79 0.77 1.09 0.97 0.89 Y 343 0.82 0.91 0.81 0.90 0.89 0.98 349 0.70 0.53 0.70 1.31 1.31 1.00 Y 351 0.68 1.04 0.66 0.66 0.63 0.96 y 353 0.67 1.21 1.04 0.55 0.86 1.56 362 0.80 0.86 0.98 0.92 1.14 1.23 363 0.80 0.96 0.98 0.84 1.02 1.22 Y 364 0.84 0.80 0.77 1.05 0.97 0.92 Y 365 0.92 0.74 0.94 1.25 1.27 1.01 367 0.64 0.71 0.54 0.89 0.75 0.84 Y 369 0.92 0.37 0.40 2.49 1.08 0.43 Y 371 0.88 0.88 0.93 1.00 1.06 1.06 372 0.76 0.82 0.86 0.93 1.05 1.12 373 0.98 0.79 0.87 1.25 1.11 0.89 375 0.84 0.77 0.92 1.08 1.19 1.11 379 0.82 0.80 0.78 1.02 0.97 0.95 380 0.82 0.78 0.71 1.05 0.92 0.88 381 0.90 0.86 0.77 1.06 0.90 0.85 382 0.95 0.84 0.52 1.13 0.62 0.55 Y 383 0.82 0.87 0.93 0.94 1.06 1.13 386 1.07 0.89 0.93 1.21 1.05 0.87 387 0.84 0.86 0.80 0.98 0.93 0.96 392 1.01 0.75 0.40 1.35 0.54 0.40 Y 397 0.86 0.93 0.91 0.92 0.97 1.06 Y 399 0.81 0.67 0.69 1.22 1.03 0.85 Y 405 0.68 0.70 0.65 0.97 0.93 0.96 410 0.91 0.88 1.00 1.03 1.13 1.09 411 0.88 0.82 0.83 1.07 1.01 0.94 Y 412 0.88 0.83 0.93 1.06 1.12 1.06 418 0.91 0.99 0.93 0.93 0.94 1.01 421 0.71 0.77 0.73 0.93 0.94 1.02 y 426 0.68 0.74 0.70 0.92 0.95 1.03 429 0.92 0.79 0.84 1.16 1.06 0.92 430 1.16 0.94 1.10 1.23 1.17 0.95 436 1.01 0.68 0.97 1.47 1.41 0.96 Y 438 0.72 0.72 0.72 1.00 1.00 1.00 439 0.72 0.83 0.82 0.87 0.98 1.13 y 442 0.75 1.06 0.66 0.71 0.62 0.88 443 0.84 0.87 0.83 0.96 0.95 0.99 445 1.10 0.21 1.00 5.28 4.83 0.92 446 0.77 0.82 0.84 0.94 1.03 1.10 Y 447 0.66 1.50 0.46 0.44 0.31 0.70 Y 451 0.74 0.65 0.63 1.13 0.97 0.86 Y 454 0.95 1.37 2.06 0.69 1.51 2.18 456 0.56 0.72 0.92 0.77 1.29 1.66 Y 459 1.35 0.13 0.96 10.23 7.30 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1.05 0.93 y 1542 0.95 0.90 0.95 1.06 1.05 0.99 1547 0.82 1.05 3.32 0.79 3.16 4.03 Y 1565 0.96 0.35 0.77 2.77 2.23 0.81 1567 1.02 0.70 0.93 1.45 1.32 0.91 1582 1.08 0.95 0.97 1.14 1.02 0.90 1711 0.69 0.40 0.64 1.75 1.61 0.92 1718 0.78 0.83 0.72 0.95 0.87 0.92 Y 1721 0.88 0.95 0.92 0.93 0.98 1.05 1731 0.63 0.79 0.63 0.79 0.80 1.01 1732 0.47 0.73 0.47 0.64 0.65 1.00 y 1744 0.79 0.84 0.79 0.94 0.94 1.00 y 1753 0.74 0.64 0.60 1.15 0.93 0.81 Y 1754 1.70 0.72 0.90 2.35 1.24 0.53 Y 1782 0.70 0.83 0.72 0.84 0.86 1.03 Y 1783 0.92 0.95 0.97 0.97 1.02 1.05 1784 0.94 0.66 0.77 1.43 1.18 0.82 Y 1786 1.02 0.76 0.94 1.34 1.24 0.92 Y 1788 0.88 0.82 0.80 1.08 0.98 0.91 Y 1789 0.87 0.80 0.82 1.08 1.02 0.95 Y 1790 0.95 0.83 0.89 1.14 1.07 0.94 Y 1791 0.83 0.81 0.75 1.02 0.93 0.91 y 1792 0.84 0.87 0.74 0.96 0.84 0.88 Y 1793 0.38 0.08 0.04 4.61 0.48 0.10 Y 1811 0.93 0.88 0.86 1.07 0.99 0.93 y 1815 0.82 0.78 0.80 1.05 1.03 0.98 y 1827 0.91 0.86 0.88 1.05 1.02 0.97 Y 1828 0.95 0.93 0.90 1.03 0.97 0.95 1829 0.87 0.73 0.79 1.18 1.07 0.91 Y 1831 0.91 0.87 0.88 1.05 1.01 0.96 Y 1834 0.91 0.99 0.99 0.92 1.00 1.09 1837 0.83 0.93 0.96 0.89 1.04 1.16 1841 0.82 0.97 0.93 0.84 0.96 1.14 y 1845 0.89 0.82 0.84 1.08 1.02 0.95 Y 1851 0.76 0.78 0.66 0.97 0.85 0.87 Y 1866 0.66 0.56 0.44 1.19 0.79 0.66 Y 1867 0.88 0.84 0.79 1.04 0.93 0.90 Y 1870 0.85 0.94 0.99 0.90 1.05 1.16 1878 0.86 0.82 0.77 1.04 0.93 0.90 Y 1884 0.79 0.83 0.69 0.95 0.84 0.88 Y 1889 0.97 1.01 0.91 0.95 0.90 0.95 1892 0.81 1.11 1.17 0.73 1.06 1.46 Y 1902 0.75 0.86 0.67 0.86 0.77 0.89 Y 1903 0.54 0.93 0.94 0.59 1.02 1.73 Y 1907 0.78 1.00 0.99 0.78 1.00 1.28 1945 0.90 0.90 0.85 1.00 0.95 0.95 y 1948 0.86 0.78 0.76 1.10 0.98 0.89 Y 1949 0.88 0.91 0.82 0.97 0.90 0.94 Y 1950 0.89 1.10 0.99 0.81 0.90 1.12 1953 0.83 0.92 0.86 0.91 0.93 1.03 1955 0.85 0.94 0.74 0.90 0.79 0.88 1962 0.84 0.85 0.75 0.99 0.89 0.89 Y 1970 0.85 0.90 0.81 0.95 0.90 0.94 y 1974 0.80 0.88 0.69 0.90 0.79 0.87 Y 1975 0.89 1.05 0.94 0.85 0.89 1.05 1976 0.77 1.18 1.06 0.66 0.90 1.38 1979 0.87 1.06 0.95 0.82 0.90 1.09 1982 0.86 0.99 0.76 0.88 0.77 0.87 1985 0.74 1.15 1.06 0.65 0.92 1.42 2016 0.90 0.93 0.92 0.97 0.99 1.02 2021 0.92 0.91 0.88 1.00 0.97 0.97 2024 0.86 0.91 0.85 0.94 0.93 0.99 2034 0.84 0.72 0.73 1.17 1.01 0.86 Y 2052 0.77 0.83 0.71 0.93 0.85 0.91 Y 2067 0.80 0.83 0.77 0.96 0.92 0.96 Y 2072 0.76 0.91 0.74 0.84 0.81 0.97 y 2105 0.83 0.56 0.65 1.49 1.16 0.78 2106 0.91 0.61 0.76 1.50 1.25 0.83 y 2107 0.88 0.65 0.75 1.36 1.17 0.86 2113 0.89 0.56 0.71 1.60 1.28 0.80 2115 0.88 0.61 0.68 1.45 1.12 0.77 2118 0.86 0.85 0.94 1.01 1.11 1.10 2136 0.49 0.75 0.39 0.65 0.51 0.78 2157 0.89 0.61 0.72 1.45 1.18 0.81 2164 0.80 0.70 0.68 1.15 0.97 0.85 Y 2165 0.60 0.91 0.64 0.67 0.70 1.05 2166 0.96 0.75 0.84 1.27 1.11 0.87 2168 0.91 0.84 0.99 1.08 1.18 1.09 2179 0.88 0.57 0.92 1.53 1.60 1.04 2192 0.73 0.50 0.69 1.46 1.38 0.95 2193 0.81 0.38 0.71 2.12 1.87 0.88 y 2194 0.66 0.76 0.59 0.87 0.78 0.90 2199 0.43 0.82 0.37 0.53 0.45 0.85 y 2203 0.60 0.98 0.64 0.61 0.65 1.07 2206 0.61 0.59 0.52 1.04 0.87 0.84 2209 0.60 0.63 0.53 0.96 0.85 0.88 y 2211 0.74 0.61 0.62 1.22 1.02 0.84 2217 0.56 0.91 0.51 0.61 0.56 0.92 y 2310 0.88 1.04 0.90 0.85 0.86 1.02 2311 0.84 1.23 1.01 0.69 0.83 1.20 2312 0.77 0.73 0.67 1.05 0.91 0.87 Y 2313 0.87 0.81 0.75 1.08 0.93 0.86 2314 0.82 0.99 0.88 0.83 0.89 1.07 2318 0.84 1.01 0.81 0.84 0.81 0.97 2327 0.80 0.92 0.82 0.87 0.89 1.03 2343 0.77 0.73 0.65 1.05 0.90 0.85 Y 2345 0.79 0.91 0.70 0.87 0.77 0.88 Y 2347 0.76 0.75 0.63 1.01 0.84 0.83 Y 2348 0.89 1.16 0.97 0.77 0.84 1.09 2350 0.84 0.85 0.85 0.99 1.00 1.01 2351 0.64 0.79 0.68 0.81 0.86 1.06 y 2356 0.76 0.66 0.77 1.14 1.17 1.02 Y 2357 0.72 0.91 0.80 0.78 0.88 1.12 2360 0.71 0.94 0.69 0.76 0.74 0.97 2361 0.61 1.00 0.75 0.61 0.75 1.24 2364 0.74 0.95 0.76 0.77 0.80 1.03 2371 0.50 1.02 0.74 0.49 0.73 1.49 2374 0.44 1.03 0.58 0.43 0.57 1.34 2375 0.75 0.80 0.73 0.94 0.91 0.98 2376 0.60 0.74 0.56 0.81 0.76 0.94 2379 0.74 0.81 0.65 0.91 0.81 0.89 2381 0.44 0.81 0.70 0.54 0.86 1.59 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1.19 0.80 0.68 Y 2499 0.72 0.71 0.67 1.02 0.95 0.93 Y 2513 0.78 0.90 0.77 0.87 0.86 0.99 Y 2514 0.73 0.63 0.56 1.16 0.88 0.76 Y 2517 0.80 0.84 0.71 0.95 0.85 0.89 Y 2518 0.70 0.75 0.72 0.93 0.96 1.03 Y 2520 0.87 0.94 0.89 0.92 0.95 1.03 2522 0.95 1.08 1.11 0.88 1.02 1.16 2523 0.88 0.86 0.82 1.01 0.95 0.93 2525 0.87 0.88 0.95 0.99 1.08 1.09 2543 0.90 0.94 0.94 0.95 1.00 1.05 2547 0.75 0.90 0.99 0.83 1.10 1.33 2549 0.98 0.90 1.22 1.08 1.35 1.25 2561 0.81 0.82 0.82 0.98 0.99 1.01 y 2563 0.84 0.67 0.70 1.24 1.03 0.83 Y 2564 0.55 0.40 0.39 1.38 0.98 0.71 Y 2565 0.98 0.93 0.99 1.05 1.06 1.01 2566 0.86 0.84 0.88 1.03 1.05 1.02 2567 0.96 0.84 0.84 1.15 1.01 0.87 y 2568 0.80 0.64 0.60 1.25 0.95 0.76 Y 2569 0.67 0.53 0.71 1.26 1.33 1.06 y 2570 0.80 0.64 0.73 1.25 1.15 0.92 Y 2575 0.96 0.87 0.98 1.10 1.12 1.02 2577 0.96 0.75 0.87 1.28 1.15 0.90 2579 0.88 0.80 0.91 1.10 1.14 1.04 2580 0.77 0.55 0.62 1.39 1.13 0.81 Y 2581 0.91 0.75 0.82 1.21 1.09 0.90 2583 0.84 0.68 0.69 1.24 1.02 0.83 Y 2588 0.92 0.84 0.88 1.09 1.04 0.95 2591 0.91 0.75 0.70 1.21 0.93 0.77 Y 2592 0.80 0.81 0.94 0.98 1.16 1.18 Y 2594 0.88 0.84 0.82 1.05 0.98 0.94 2610 0.73 0.52 0.57 1.40 1.11 0.79 Y 2624 0.91 0.78 0.73 1.17 0.94 0.80 Y 2625 0.86 0.78 0.72 1.11 0.93 0.84 Y 2649 1.10 0.66 0.97 1.67 1.46 0.87 Y 2652 0.80 0.65 0.57 1.22 0.87 0.71 Y 2669 1.20 0.11 0.74 10.96 6.75 0.62 3453 0.51 0.57 0.43 0.90 0.76 0.85 Y 3454 0.90 0.90 0.82 1.00 0.91 0.91 Y 3455 0.81 0.80 0.71 1.01 0.89 0.88 y 3456 0.56 0.45 0.31 1.24 0.69 0.56 Y 3458 0.38 0.67 0.28 0.57 0.42 0.74 Y 3461 0.83 0.69 0.73 1.20 1.05 0.88 Y 3462 0.94 0.91 0.79 1.03 0.87 0.84 Y 3464 0.84 0.46 0.69 1.81 1.49 0.83 Y 3466 0.72 0.60 0.57 1.19 0.95 0.80 Y 3471 1.03 0.77 1.07 1.35 1.40 1.04 y 3472 1.01 0.98 1.07 1.03 1.10 1.07 3473 1.16 0.78 0.92 1.49 1.18 0.79 y 3474 1.15 0.83 1.05 1.40 1.27 0.91 Y 3478 0.73 0.75 0.69 0.97 0.91 0.94 Y 3479 0.85 0.86 1.06 0.98 1.23 1.25 3480 0.89 1.01 0.98 0.88 0.97 1.10 3485 0.70 0.61 0.61 1.16 1.00 0.87 Y 3495 0.87 0.78 0.77 1.12 0.99 0.88 Y 3498 1.05 1.18 1.17 0.89 1.00 1.12 3505 0.65 0.73 0.53 0.89 0.72 0.81 Y 3507 0.83 0.73 0.76 1.13 1.04 0.92 Y 3508 0.83 0.90 0.82 0.92 0.91 0.98 Y 3513 0.83 1.05 0.89 0.79 0.84 1.07 3514 0.73 1.07 0.82 0.69 0.76 1.11 3516 0.74 0.69 0.68 1.07 0.98 0.91 Y 3522 0.76 0.58 0.68 1.32 1.17 0.89 Y 3528 0.84 0.74 0.64 1.15 0.87 0.76 y 3530 0.91 0.94 0.88 0.98 0.94 0.96 3533 1.11 1.07 1.58 1.04 1.48 1.42 Y 3534 0.97 0.89 1.88 1.08 2.10 1.94 Y 3535 0.99 0.81 0.94 1.22 1.15 0.95 3537 0.81 0.77 0.81 1.05 1.05 1.00 y 3541 0.58 0.81 0.55 0.72 0.68 0.95 3542 0.97 0.99 1.11 0.98 1.12 1.14 3547 0.82 0.86 0.74 0.95 0.86 0.90 Y 3548 0.68 0.82 0.61 0.82 0.74 0.90 Y 3549 0.97 0.80 0.85 1.21 1.06 0.88 Y 3550 0.91 0.73 0.71 1.26 0.98 0.78 Y 3552 1.14 0.91 0.73 1.25 0.80 0.64 Y 3563 1.07 0.78 0.86 1.37 1.11 0.81 3565 0.74 0.63 0.76 1.18 1.20 1.02 3566 0.79 0.64 0.62 1.23 0.98 0.79 Y 3567 0.83 0.74 0.75 1.13 1.02 0.90 Y 3568 0.84 0.79 1.03 1.06 1.31 1.23 Y 3572 0.58 0.81 0.65 0.72 0.81 1.12 3573 0.90 0.98 1.06 0.92 1.09 1.18 3576 0.64 0.65 0.63 0.99 0.97 0.98 Y 3577 0.77 0.84 0.73 0.92 0.87 0.95 Y 3578 0.86 0.90 1.01 0.96 1.12 1.17 3579 0.54 0.50 0.45 1.07 0.90 0.84 Y 3580 0.82 0.72 0.75 1.14 1.04 0.92 y 3585 0.76 0.93 0.89 0.81 0.95 1.17 3587 0.59 0.68 0.54 0.86 0.80 0.93 Y 3588 0.80 0.73 0.72 1.10 0.98 0.89 Y 3589 0.46 0.35 0.36 1.31 1.04 0.79 Y 3598 0.72 0.79 0.64 0.91 0.82 0.89 Y 3599 1.09 0.81 0.96 1.35 1.18 0.88 3602 0.68 0.91 0.80 0.74 0.87 1.18 3603 0.67 0.74 0.75 0.91 1.02 1.12 Y 3607 0.49 1.11 0.44 0.44 0.40 0.90 Y 3608 0.56 1.00 0.61 0.56 0.61 1.08 Y 3624 0.84 0.85 0.84 0.99 0.99 1.00 Y 3627 0.88 0.70 0.72 1.26 1.03 0.82 3628 0.85 0.85 0.88 1.00 1.04 1.04 3636 1.58 1.35 1.41 1.17 1.04 0.89 Y 3641 0.90 0.58 0.55 1.55 0.95 0.61 Y 3643 1.59 1.08 0.93 1.48 0.86 0.58 Y 3644 1.65 1.04 0.97 1.58 0.94 0.59 Y 3648 0.89 0.95 0.88 0.94 0.92 0.98 3649 0.87 0.77 0.77 1.13 1.00 0.88 Y 3650 0.77 0.85 0.71 0.91 0.83 0.92 3651 0.98 0.80 0.72 1.22 0.90 0.74 Y 3653 0.65 0.97 0.69 0.68 0.71 1.05 Y 3654 0.84 0.82 0.65 1.03 0.79 0.77 Y 3656 0.81 0.94 0.84 0.87 0.89 1.03 3658 0.65 0.78 0.57 0.84 0.74 0.88 y 3662 0.67 1.07 0.60 0.63 0.57 0.91 3663 0.85 0.24 0.66 3.56 2.77 0.78 3664 1.08 0.51 1.20 2.10 2.35 1.12 y 3667 1.03 0.61 0.99 1.68 1.62 0.96 3668 0.80 0.74 0.78 1.07 1.05 0.98 Y 3670 0.61 1.31 0.90 0.46 0.69 1.49 3694 0.81 0.37 0.44 2.17 1.17 0.54 Y 3698 1.12 0.92 0.80 1.22 0.87 0.71 3714 0.42 0.89 0.41 0.47 0.46 0.97 Y 3715 0.53 0.78 0.66 0.68 0.84 1.25 y 3718 0.66 0.92 0.71 0.72 0.78 1.08 3727 0.64 0.67 0.56 0.96 0.84 0.88 Y 3728 0.89 0.84 0.83 1.06 0.99 0.93 3751 0.81 0.95 0.58 0.85 0.61 0.72 Y 3754 0.89 0.83 0.72 1.07 0.87 0.81 Y 3757 0.42 0.24 0.24 1.72 1.00 0.58 Y 3771 0.92 0.96 1.12 0.96 1.17 1.22 3978 0.84 0.59 0.81 1.43 1.37 0.96 4000 1.01 0.67 1.24 1.52 1.86 1.22 y 4079 0.98 1.19 0.79 0.83 0.66 0.80 Y 4108 0.72 0.38 0.46 1.91 1.24 0.65 Y 4131 0.88 0.94 1.00 0.93 1.07 1.14 4144 0.95 0.75 0.84 1.26 1.12 0.88 y 4221 0.77 0.44 0.57 1.75 1.28 0.73 Y 4246 1.45 0.62 0.98 2.32 1.57 0.68 Y 4274 0.98 0.46 0.40 2.14 0.87 0.41 Y 4363 1.03 0.56 0.78 1.84 1.39 0.76 4756 0.92 0.71 0.74 1.29 1.04 0.80 4832 0.95 1.06 0.96 0.90 0.91 1.02 4835 0.95 0.47 0.58 2.01 1.22 0.61 Y 4899 1.68 0.81 0.62 2.09 0.77 0.37 Y 4926 0.91 0.91 18.88 1.00 20.85 20.85 Y 4930 2.31 1.16 1.25 1.99 1.07 0.54 Y 4971 0.47 1.08 0.56 0.44 0.52 1.19

TABLE 4D Sp/SPF ABX/SPF GF/SPF PSO p-value q-value p-value q-value p-value q-value 1 0.0548 0.0477 0.0416 0.0281 0.5657 0.2280 2 0.5443 0.2032 0.9539 0.2475 0.1960 0.1011 5 0.0091 0.0213 0.0012 0.0040 0.0690 0.0459 6 0.0331 0.0377 0.0003 0.0022 0.0297 0.0247 9 0.2191 0.1076 0.4977 0.1556 0.6786 0.2604 10 0.0084 0.0209 0.0133 0.0134 0.1705 0.0922 14 0.4825 0.1882 0.7326 0.2040 0.0829 0.0532 16 0.0387 0.0399 0.0549 0.0335 0.3766 0.1644 17 0.2213 0.1081 0.0664 0.0374 0.9339 0.3236 18 0.4364 0.1725 0.1559 0.0679 0.4767 0.1979 21 0.0088 0.0212 0.0011 0.0040 0.0114 0.0136 28 0.0245 0.0336 0.0321 0.0238 0.0642 0.0430 30 0.0167 0.0286 0.0003 0.0022 0.0277 0.0239 34 0.0155 0.0279 0.0118 0.0128 0.0201 0.0194 35 0.0443 0.0428 0.0034 0.0064 0.0098 0.0118 38 0.7219 0.2444 0.7369 0.2044 0.7925 0.2902 39 0.7490 0.2502 0.4928 0.1543 0.9183 0.3196 40 0.0816 0.0595 0.0044 0.0068 0.0056 0.0083 41 0.0264 0.0342 0.0075 0.0095 0.0229 0.0214 42 0.0903 0.0627 0.0010 0.0037 0.0003 0.0015 43 0.0006 0.0059 0.1065 0.0509 0.0061 0.0089 44 0.0287 0.0345 0.0218 0.0186 0.0291 0.0244 45 0.0628 0.0518 0.0020 0.0053 0.0009 0.0028 47 0.0001 0.0027 0.7104 0.2003 0.6574 0.2548 50 0.1912 0.0992 0.0265 0.0216 0.2104 0.1066 51 0.9870 0.3040 0.0347 0.0252 0.0001 0.0006 52 0.0932 0.0637 0.0362 0.0260 0.0068 0.0095 53 0.0239 0.0336 0.0056 0.0078 0.0007 0.0024 54 0.0188 0.0303 0.0044 0.0068 0.0013 0.0034 55 0.3565 0.1494 0.5096 0.1583 0.0636 0.0427 56 0.4956 0.1903 0.2803 0.1053 0.2762 0.1323 59 0.7848 0.2596 0.0683 0.0379 0.4103 0.1738 65 0.6905 0.2378 0.2604 0.0996 0.6012 0.2402 66 0.1604 0.0917 0.0568 0.0340 0.0824 0.0532 67 0.2818 0.1273 0.8419 0.2253 0.1039 0.0620 68 0.0272 0.0345 0.7633 0.2109 0.9673 0.3316 74 0.0121 0.0243 0.0000 0.0001 0.0000 0.0001 75 0.6028 0.2164 0.0170 0.0160 0.1938 0.1008 76 0.1781 0.0958 0.0119 0.0128 0.0017 0.0038 77 0.0140 0.0262 0.0074 0.0095 0.0179 0.0181 80 0.5221 0.1970 0.0042 0.0068 0.0025 0.0052 82 0.2101 0.1043 0.0282 0.0222 0.0321 0.0263 83 0.1343 0.0794 0.0130 0.0134 0.0286 0.0241 84 0.0083 0.0209 0.0441 0.0290 0.0181 0.0182 92 0.0650 0.0521 0.1027 0.0494 0.3484 0.1549 93 0.3313 0.1431 0.0010 0.0038 0.0049 0.0076 99 0.1851 0.0975 0.0010 0.0037 0.0001 0.0006 100 0.0973 0.0650 0.0029 0.0060 0.0025 0.0051 101 0.0311 0.0364 0.0022 0.0053 0.0064 0.0092 102 0.0016 0.0108 0.0450 0.0294 0.0020 0.0043 103 0.0304 0.0358 0.0048 0.0070 0.0259 0.0229 105 0.5712 0.2107 0.6866 0.1972 0.9818 0.3354 107 0.9162 0.2896 0.6989 0.1994 0.4234 0.1784 113 0.1014 0.0665 0.0036 0.0064 0.0001 0.0010 114 0.6511 0.2261 0.9454 0.2458 0.0955 0.0583 117 0.6489 0.2261 0.7412 0.2052 0.2420 0.1197 119 0.0019 0.0111 0.0000 0.0000 0.0000 0.0000 120 0.1662 0.0923 0.0256 0.0213 0.0087 0.0109 121 0.9196 0.2899 0.2821 0.1053 0.1915 0.1003 122 0.6361 0.2231 0.2334 0.0912 0.2963 0.1388 126 0.2086 0.1039 0.0302 0.0231 0.0242 0.0223 137 0.5909 0.2140 0.2049 0.0843 0.0185 0.0184 138 0.3525 0.1484 0.1922 0.0805 0.0027 0.0053 150 0.8465 0.2728 0.0460 0.0300 0.0199 0.0193 154 0.1993 0.1020 0.0115 0.0126 0.0115 0.0136 180 0.4578 0.1794 0.6131 0.1813 0.7601 0.2840 181 0.3765 0.1552 0.5436 0.1663 0.8418 0.3054 191 0.9835 0.3040 0.1710 0.0733 0.8645 0.3090 210 0.0070 0.0206 0.0132 0.0134 0.0034 0.0059 217 0.0075 0.0209 0.0005 0.0025 0.0012 0.0033 221 0.3849 0.1568 0.2091 0.0856 0.0576 0.0398 227 0.9087 0.2891 0.0715 0.0387 0.7673 0.2859 241 0.2545 0.1190 0.0215 0.0185 0.0593 0.0404 254 0.0326 0.0377 0.0000 0.0005 0.0000 0.0000 261 0.0657 0.0521 0.0277 0.0221 0.0178 0.0181 263 0.8071 0.2640 0.2960 0.1084 0.8947 0.3140 265 0.1993 0.1020 0.2448 0.0944 0.5508 0.2238 273 0.6969 0.2387 0.4676 0.1503 0.3594 0.1577 280 0.0017 0.0110 0.1725 0.0737 0.0246 0.0224 296 0.1239 0.0764 0.0076 0.0096 0.0102 0.0122 299 0.5620 0.2085 0.5203 0.1602 0.4476 0.1864 300 0.1808 0.0961 0.8696 0.2306 0.4480 0.1864 301 0.0001 0.0027 0.0530 0.0331 0.0015 0.0037 303 0.7050 0.2403 0.4495 0.1471 0.7707 0.2864 306 0.2561 0.1190 0.2443 0.0944 0.3871 0.1679 311 0.1138 0.0721 0.0000 0.0004 0.0056 0.0083 312 0.0053 0.0186 0.0013 0.0041 0.0261 0.0230 318 0.2549 0.1190 0.1778 0.0754 0.3973 0.1712 321 0.7147 0.2424 0.0173 0.0161 0.3452 0.1542 323 0.2006 0.1020 0.3965 0.1348 0.3583 0.1577 326 0.8307 0.2693 0.8387 0.2249 0.2531 0.1233 328 0.1707 0.0927 0.0695 0.0382 0.8619 0.3088 332 0.0001 0.0027 0.0000 0.0001 0.0000 0.0002 333 0.7058 0.2403 0.0006 0.0029 0.1961 0.1011 334 0.0211 0.0316 0.0908 0.0447 0.0492 0.0356 337 0.0590 0.0504 0.0000 0.0005 0.0536 0.0374 342 0.0399 0.0407 0.0021 0.0053 0.0008 0.0026 343 0.2256 0.1086 0.6827 0.1968 0.1918 0.1003 349 0.0381 0.0398 0.0012 0.0040 0.0270 0.0234 351 0.0860 0.0614 0.7037 0.1994 0.0583 0.0402 353 0.3788 0.1558 0.5034 0.1566 0.8886 0.3131 362 0.2909 0.1299 0.4077 0.1371 0.9112 0.3186 363 0.0071 0.0206 0.5988 0.1787 0.7948 0.2905 364 0.0104 0.0222 0.0019 0.0050 0.0005 0.0022 365 0.4673 0.1827 0.0249 0.0208 0.6053 0.2406 367 0.0296 0.0351 0.1076 0.0511 0.0040 0.0066 369 0.5338 0.2006 0.0000 0.0000 0.0000 0.0001 371 0.0869 0.0616 0.0806 0.0420 0.3378 0.1526 372 0.1303 0.0780 0.2841 0.1053 0.4466 0.1864 373 0.9750 0.3020 0.1934 0.0807 0.4323 0.1814 375 0.2567 0.1190 0.1054 0.0505 0.6188 0.2452 379 0.0693 0.0535 0.0548 0.0335 0.0373 0.0294 380 0.1268 0.0773 0.0793 0.0414 0.0176 0.0180 381 0.3829 0.1563 0.1361 0.0611 0.0191 0.0188 382 0.7542 0.2514 0.2125 0.0861 0.0000 0.0003 383 0.1610 0.0917 0.3459 0.1223 0.6412 0.2520 386 0.5064 0.1936 0.1941 0.0808 0.3594 0.1577 387 0.0810 0.0593 0.1523 0.0667 0.0508 0.0366 392 0.5783 0.2127 0.3183 0.1152 0.0002 0.0014 397 0.0039 0.0164 0.1396 0.0619 0.0523 0.0371 399 0.1296 0.0780 0.0046 0.0069 0.0082 0.0108 405 0.1108 0.0713 0.1095 0.0513 0.0484 0.0353 410 0.2713 0.1242 0.1277 0.0577 0.9608 0.3309 411 0.0330 0.0377 0.0028 0.0059 0.0039 0.0065 412 0.1453 0.0850 0.0390 0.0271 0.3557 0.1571 418 0.2303 0.1103 0.8272 0.2228 0.3077 0.1424 421 0.0230 0.0326 0.0705 0.0385 0.0402 0.0309 426 0.0270 0.0345 0.0755 0.0401 0.0473 0.0347 429 0.3171 0.1389 0.0397 0.0275 0.1222 0.0698 430 0.1178 0.0736 0.4832 0.1523 0.2921 0.1376 436 0.8713 0.2798 0.0040 0.0065 0.6503 0.2531 438 0.1247 0.0764 0.1218 0.0557 0.1437 0.0795 439 0.0073 0.0209 0.1116 0.0520 0.0862 0.0547 442 0.3193 0.1395 0.6039 0.1795 0.1953 0.1011 443 0.0373 0.0394 0.1022 0.0493 0.0326 0.0265 445 0.8455 0.2728 0.0907 0.0447 0.7607 0.2840 446 0.0053 0.0186 0.0323 0.0239 0.0711 0.0471 447 0.2880 0.1291 0.0659 0.0373 0.0518 0.0369 451 0.0141 0.0262 0.0016 0.0046 0.0007 0.0025 454 0.9387 0.2935 0.4996 0.1558 0.1163 0.0676 456 0.0039 0.0164 0.0880 0.0441 0.3507 0.1552 459 0.5363 0.2011 0.0261 0.0214 0.6215 0.2458 460 0.6506 0.2261 0.4757 0.1514 0.0551 0.0383 461 0.9395 0.2935 0.4258 0.1416 0.8906 0.3131 465 0.0159 0.0282 0.4488 0.1471 0.0853 0.0543 467 0.0016 0.0108 0.0623 0.0356 0.0005 0.0022 468 0.0084 0.0209 0.0848 0.0430 0.0012 0.0033 469 0.0037 0.0164 0.2144 0.0861 0.0361 0.0288 471 0.3007 0.1331 0.3236 0.1158 0.0192 0.0188 472 0.1667 0.0923 0.0533 0.0331 0.1041 0.0620 473 0.5885 0.2138 0.0817 0.0423 0.5027 0.2078 474 0.0174 0.0289 0.0004 0.0025 0.0123 0.0144 475 0.0411 0.0412 0.4510 0.1473 0.0372 0.0294 476 0.8311 0.2693 0.2204 0.0880 0.3746 0.1640 477 0.0375 0.0394 0.0332 0.0243 0.0585 0.0402 478 0.0015 0.0108 0.0764 0.0404 0.0030 0.0056 479 0.3103 0.1366 0.0275 0.0221 0.0150 0.0161 480 0.3659 0.1522 0.3202 0.1152 0.2309 0.1148 756 0.3654 0.1522 0.0271 0.0218 0.0923 0.0569 929 0.5433 0.2032 0.0079 0.0096 0.0079 0.0107 955 0.0098 0.0218 0.2877 0.1058 0.0012 0.0033 958 0.3663 0.1522 0.1561 0.0679 0.1130 0.0660 959 0.1689 0.0924 0.0053 0.0075 0.1106 0.0654 965 0.2470 0.1167 0.0022 0.0053 0.1838 0.0969 967 0.0286 0.0345 0.0079 0.0096 0.0032 0.0057 972 0.7869 0.2599 0.3870 0.1326 0.5267 0.2158 973 0.8061 0.2640 0.4549 0.1476 0.3339 0.1517 974 0.0447 0.0428 0.0129 0.0134 0.1791 0.0949 975 0.0005 0.0056 0.0001 0.0005 0.0010 0.0032 978 0.9885 0.3040 0.1867 0.0786 0.6942 0.2643 981 0.0542 0.0477 0.2267 0.0893 0.6479 0.2526 982 0.4432 0.1744 0.0189 0.0171 0.4315 0.1814 983 0.9472 0.2949 0.8998 0.2365 0.6677 0.2567 984 0.3448 0.1469 0.3587 0.1256 0.4059 0.1733 986 0.0624 0.0518 0.0033 0.0064 0.0320 0.0262 993 0.0148 0.0272 0.4881 0.1535 0.2286 0.1140 994 0.0012 0.0093 0.0003 0.0021 0.0457 0.0339 995 0.0638 0.0521 0.1599 0.0694 0.2922 0.1376 997 0.0215 0.0318 0.0158 0.0152 0.0053 0.0081 1018 0.0900 0.0627 0.0000 0.0001 0.0047 0.0075 1020 0.0287 0.0345 0.0035 0.0064 0.0170 0.0174 1023 0.0217 0.0319 0.0002 0.0018 0.0049 0.0076 1024 0.0027 0.0145 0.0472 0.0305 0.0085 0.0109 1028 0.1334 0.0791 0.0128 0.0134 0.0090 0.0111 1030 0.0753 0.0566 0.0030 0.0060 0.0086 0.0109 1033 0.1458 0.0850 0.0289 0.0222 0.1117 0.0658 1073 0.4055 0.1633 0.0188 0.0171 0.9752 0.3338 1078 0.0016 0.0108 0.0001 0.0005 0.0001 0.0011 1079 0.0262 0.0342 0.0095 0.0111 0.2445 0.1201 1080 0.2055 0.1030 0.0061 0.0083 0.1997 0.1020 1090 0.1167 0.0732 0.0096 0.0111 0.1746 0.0941 1099 0.0249 0.0338 0.0150 0.0149 0.0128 0.0148 1104 0.1581 0.0910 0.0287 0.0222 0.1761 0.0942 1108 0.0419 0.0418 0.0133 0.0134 0.1971 0.1012 1109 0.6916 0.2378 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0.8625 0.3088 1203 0.1969 0.1019 0.6710 0.1942 0.8455 0.3061 1204 0.5120 0.1952 0.1269 0.0577 0.1178 0.0681 1205 0.3455 0.1469 0.6358 0.1859 0.3340 0.1517 1217 0.0487 0.0447 0.5537 0.1687 0.0778 0.0511 1218 0.0081 0.0209 0.2226 0.0884 0.0021 0.0044 1223 0.0920 0.0633 0.5789 0.1745 0.2451 0.1201 1225 0.0182 0.0297 0.4807 0.1520 0.0185 0.0184 1236 0.0501 0.0455 0.6842 0.1969 0.3370 0.1526 1239 0.0940 0.0641 0.2318 0.0908 0.0870 0.0549 1244 0.1143 0.0722 0.9588 0.2475 0.3050 0.1415 1247 0.0625 0.0518 0.2825 0.1053 0.0204 0.0196 1253 0.0042 0.0169 0.1081 0.0511 0.0397 0.0307 1258 0.0044 0.0169 0.9563 0.2475 0.0011 0.0032 1259 0.0197 0.0311 0.7007 0.1994 0.0100 0.0121 1260 0.0178 0.0293 0.3749 0.1300 0.0074 0.0101 1264 0.1640 0.0923 0.7715 0.2128 0.0881 0.0552 1265 0.0063 0.0199 0.4549 0.1476 0.0232 0.0215 1267 0.1873 0.0980 0.7023 0.1994 0.0899 0.0560 1269 0.0071 0.0206 0.5167 0.1594 0.0168 0.0173 1270 0.0212 0.0317 0.4066 0.1371 0.0149 0.0161 1271 0.2503 0.1176 0.7887 0.2163 0.1964 0.1011 1272 0.0850 0.0610 0.4064 0.1371 0.0909 0.0564 1273 0.0286 0.0345 0.2812 0.1053 0.1121 0.0658 1274 0.0260 0.0342 0.4067 0.1371 0.0465 0.0343 1276 0.0764 0.0567 0.9658 0.2488 0.0825 0.0532 1277 0.0949 0.0642 0.5427 0.1663 0.1451 0.0800 1351 0.7712 0.2561 0.0131 0.0134 0.1758 0.0942 1355 0.0084 0.0209 0.0049 0.0071 0.0128 0.0148 1359 0.0699 0.0537 0.0477 0.0305 0.0236 0.0218 1360 0.3241 0.1413 0.2133 0.0861 0.9511 0.3284 1363 0.2205 0.1080 0.2480 0.0954 0.7531 0.2828 1372 0.1691 0.0924 0.6654 0.1930 0.8791 0.3125 1430 0.2336 0.1110 0.1155 0.0531 0.7501 0.2822 1446 0.3972 0.1611 0.3729 0.1296 0.1014 0.0608 1449 0.4257 0.1687 0.1443 0.0635 0.7732 0.2865 1452 0.6222 0.2205 0.0018 0.0050 0.2910 0.1376 1480 0.2975 0.1322 0.0096 0.0111 0.8213 0.2991 1482 0.1755 0.0947 0.2191 0.0877 0.9634 0.3309 1483 0.0839 0.0607 0.2273 0.0893 0.2445 0.1201 1485 0.0564 0.0487 0.9090 0.2379 0.0029 0.0055 1487 0.4882 0.1896 0.1128 0.0524 0.3045 0.1415 1488 0.0472 0.0435 0.9923 0.2543 0.0141 0.0156 1495 0.2233 0.1081 0.2683 0.1018 0.0081 0.0108 1496 0.1398 0.0823 0.0738 0.0393 0.1609 0.0875 1498 0.9880 0.3040 0.0047 0.0069 0.3986 0.1714 1499 0.1241 0.0764 0.0420 0.0281 0.0286 0.0241 1500 0.2032 0.1024 0.0006 0.0028 0.0143 0.0156 1501 0.2558 0.1190 0.0716 0.0387 0.0031 0.0056 1503 0.0172 0.0288 0.3248 0.1160 0.0003 0.0017 1504 0.8159 0.2659 0.0072 0.0094 0.3279 0.1496 1506 0.0715 0.0547 0.2859 0.1057 0.0022 0.0046 1517 0.7317 0.2468 0.0046 0.0069 0.0445 0.0332 1518 0.5518 0.2056 0.0733 0.0392 0.7864 0.2891 1519 0.3448 0.1469 0.0184 0.0170 0.5347 0.2182 1521 0.7746 0.2568 0.8307 0.2231 0.5747 0.2301 1522 0.9261 0.2915 0.0898 0.0447 0.1337 0.0750 1523 0.8055 0.2640 0.0315 0.0235 0.6361 0.2511 1524 0.6031 0.2164 0.0004 0.0022 0.0360 0.0288 1527 0.0665 0.0522 0.1610 0.0696 0.0940 0.0576 1528 0.2146 0.1060 0.2077 0.0853 0.0377 0.0295 1529 0.3545 0.1489 0.3191 0.1152 0.2719 0.1305 1536 0.6262 0.2210 0.1112 0.0519 0.6477 0.2526 1537 0.8225 0.2675 0.2419 0.0938 0.3417 0.1534 1539 0.2014 0.1021 0.0827 0.0426 0.0004 0.0018 1541 0.1004 0.0660 0.0157 0.0152 0.0395 0.0306 1542 0.3747 0.1548 0.0708 0.0385 0.3478 0.1549 1547 0.5716 0.2107 0.9753 0.2508 0.0327 0.0265 1565 0.9697 0.3014 0.1068 0.0509 0.7466 0.2814 1567 0.9168 0.2896 0.0633 0.0361 0.7791 0.2875 1582 0.6348 0.2231 0.9787 0.2513 0.9842 0.3356 1711 0.4223 0.1677 0.0600 0.0347 0.1683 0.0913 1718 0.0160 0.0282 0.0500 0.0315 0.0027 0.0053 1721 0.0170 0.0287 0.2792 0.1053 0.1217 0.0697 1731 0.1086 0.0705 0.1284 0.0578 0.1056 0.0627 1732 0.0286 0.0345 0.1384 0.0619 0.0282 0.0241 1744 0.0207 0.0316 0.0809 0.0420 0.0226 0.0213 1753 0.0277 0.0345 0.0025 0.0055 0.0006 0.0023 1754 0.0125 0.0248 0.3481 0.1228 0.9189 0.3196 1782 0.0003 0.0041 0.0289 0.0222 0.0007 0.0024 1783 0.1458 0.0850 0.3440 0.1219 0.5211 0.2144 1784 0.5126 0.1952 0.0039 0.0065 0.0645 0.0431 1786 0.7888 0.2600 0.0000 0.0002 0.1827 0.0966 1788 0.0463 0.0435 0.0035 0.0064 0.0017 0.0038 1789 0.0629 0.0518 0.0061 0.0083 0.0131 0.0150 1790 0.2736 0.1246 0.0007 0.0030 0.0164 0.0170 1791 0.0956 0.0642 0.0729 0.0392 0.0086 0.0109 1792 0.0243 0.0336 0.0732 0.0392 0.0004 0.0018 1793 0.0018 0.0110 0.0000 0.0000 0.0000 0.0000 1811 0.2022 0.1022 0.0224 0.0189 0.0136 0.0154 1815 0.0511 0.0462 0.0210 0.0183 0.0359 0.0288 1827 0.0410 0.0412 0.0038 0.0065 0.0095 0.0116 1828 0.2319 0.1105 0.0908 0.0447 0.0269 0.0234 1829 0.0521 0.0466 0.0003 0.0022 0.0030 0.0056 1831 0.0691 0.0535 0.0087 0.0104 0.0130 0.0150 1834 0.0912 0.0631 0.8257 0.2228 0.7553 0.2831 1837 0.0672 0.0525 0.4757 0.1514 0.6439 0.2521 1841 0.0114 0.0232 0.7067 0.1998 0.2837 0.1345 1845 0.0922 0.0633 0.0090 0.0106 0.0198 0.0193 1851 0.0139 0.0262 0.0306 0.0232 0.0006 0.0024 1866 0.0090 0.0213 0.0019 0.0050 0.0001 0.0008 1867 0.0499 0.0455 0.0160 0.0152 0.0014 0.0036 1870 0.0405 0.0411 0.4123 0.1381 0.8618 0.3088 1878 0.0375 0.0394 0.0120 0.0128 0.0013 0.0034 1884 0.0288 0.0345 0.0973 0.0471 0.0017 0.0038 1889 0.5884 0.2138 0.7778 0.2137 0.1166 0.0676 1892 0.0752 0.0566 0.3546 0.1245 0.2182 0.1094 1902 0.0538 0.0476 0.3969 0.1348 0.0084 0.0109 1903 0.0006 0.0059 0.6230 0.1829 0.5669 0.2280 1907 0.2068 0.1033 0.8675 0.2305 0.9916 0.3375 1945 0.0786 0.0581 0.0850 0.0430 0.0159 0.0168 1948 0.0559 0.0485 0.0044 0.0068 0.0022 0.0046 1949 0.0343 0.0378 0.1087 0.0511 0.0031 0.0056 1950 0.2685 0.1232 0.3529 0.1242 0.8509 0.3064 1953 0.0886 0.0623 0.4607 0.1488 0.1507 0.0824 1955 0.2317 0.1105 0.7745 0.2132 0.0378 0.0295 1962 0.0467 0.0435 0.0585 0.0344 0.0029 0.0055 1970 0.0617 0.0518 0.2124 0.0861 0.0138 0.0155 1974 0.0868 0.0616 0.4107 0.1378 0.0084 0.0109 1975 0.3281 0.1423 0.6139 0.1813 0.5231 0.2148 1976 0.1638 0.0923 0.3077 0.1118 0.8335 0.3030 1979 0.4077 0.1638 0.5491 0.1677 0.7717 0.2864 1982 0.4393 0.1733 0.8521 0.2272 0.1430 0.0793 1985 0.2107 0.1043 0.4184 0.1394 0.8870 0.3131 2016 0.2000 0.1020 0.3786 0.1307 0.2785 0.1328 2021 0.2644 0.1217 0.2679 0.1018 0.1227 0.0699 2024 0.1691 0.0924 0.4538 0.1476 0.1497 0.0821 2034 0.0629 0.0518 0.0021 0.0053 0.0027 0.0053 2052 0.0363 0.0394 0.1278 0.0577 0.0064 0.0092 2067 0.0224 0.0321 0.0593 0.0346 0.0082 0.0108 2072 0.0453 0.0429 0.5134 0.1588 0.0280 0.0240 2105 0.3666 0.1522 0.0287 0.0222 0.0529 0.0373 2106 0.5821 0.2132 0.0201 0.0177 0.1236 0.0702 2107 0.4095 0.1641 0.0375 0.0264 0.1132 0.0660 2113 0.5529 0.2056 0.0359 0.0259 0.1489 0.0819 2115 0.4946 0.1903 0.0558 0.0337 0.0822 0.0532 2118 0.1650 0.0923 0.2025 0.0838 0.5402 0.2200 2136 0.1751 0.0947 0.3200 0.1152 0.0845 0.0540 2157 0.4918 0.1901 0.0437 0.0290 0.1009 0.0607 2164 0.0747 0.0566 0.0102 0.0117 0.0034 0.0059 2165 0.1129 0.0719 0.3057 0.1116 0.1384 0.0772 2166 0.5884 0.2138 0.0952 0.0463 0.1986 0.1017 2168 0.4012 0.1623 0.2840 0.1053 0.8893 0.3131 2179 0.5866 0.2138 0.0575 0.0343 0.4381 0.1834 2192 0.2598 0.1201 0.0312 0.0235 0.2109 0.1066 2193 0.6240 0.2207 0.0152 0.0149 0.2129 0.1073 2194 0.1190 0.0741 0.0888 0.0443 0.0485 0.0353 2199 0.0448 0.0428 0.3891 0.1330 0.0138 0.0155 2203 0.1164 0.0732 0.6948 0.1988 0.2237 0.1119 2206 0.1325 0.0788 0.0645 0.0366 0.0254 0.0229 2209 0.0833 0.0605 0.0417 0.0281 0.0139 0.0155 2211 0.2750 0.1249 0.0560 0.0337 0.0920 0.0569 2217 0.0385 0.0399 0.6518 0.1894 0.0255 0.0229 2310 0.3826 0.1563 0.6344 0.1859 0.4011 0.1721 2311 0.4184 0.1665 0.2032 0.0839 0.9296 0.3227 2312 0.0036 0.0164 0.0008 0.0033 0.0001 0.0006 2313 0.4932 0.1902 0.2142 0.0861 0.0865 0.0547 2314 0.1989 0.1020 0.9039 0.2371 0.3942 0.1702 2318 0.2850 0.1284 0.8459 0.2260 0.1606 0.0875 2327 0.1595 0.0915 0.7144 0.2004 0.2137 0.1074 2343 0.0140 0.0262 0.0073 0.0094 0.0005 0.0022 2345 0.0547 0.0477 0.4353 0.1439 0.0075 0.0103 2347 0.0150 0.0273 0.0160 0.0152 0.0003 0.0014 2348 0.6456 0.2260 0.2830 0.1053 0.8884 0.3131 2350 0.1878 0.0980 0.2129 0.0861 0.2994 0.1399 2351 0.0004 0.0051 0.0402 0.0275 0.0017 0.0038 2356 0.0198 0.0311 0.0022 0.0053 0.0283 0.0241 2357 0.1659 0.0923 0.4181 0.1394 0.3269 0.1495 2360 0.2235 0.1081 0.4487 0.1471 0.2069 0.1051 2361 0.3381 0.1452 0.7991 0.2179 0.6023 0.2402 2364 0.1868 0.0980 0.5694 0.1720 0.3092 0.1428 2371 0.1001 0.0660 0.8120 0.2202 0.6437 0.2521 2374 0.0525 0.0467 0.7956 0.2177 0.2565 0.1241 2375 0.2803 0.1269 0.1729 0.0737 0.2444 0.1201 2376 0.1088 0.0705 0.1646 0.0710 0.0993 0.0601 2379 0.1843 0.0975 0.2257 0.0891 0.0827 0.0532 2381 0.0891 0.0624 0.2917 0.1070 0.6854 0.2625 2383 0.0643 0.0521 0.5668 0.1716 0.1260 0.0711 2394 0.0210 0.0316 0.3783 0.1307 0.0611 0.0415 2395 0.0038 0.0164 0.5901 0.1768 0.3208 0.1471 2407 0.0991 0.0657 0.0869 0.0437 0.3387 0.1526 2411 0.0649 0.0521 0.0837 0.0427 0.0371 0.0294 2413 0.0001 0.0027 0.0694 0.0382 0.0038 0.0064 2416 0.6176 0.2196 0.1180 0.0541 0.3773 0.1644 2418 0.0046 0.0169 0.6502 0.1894 0.3113 0.1434 2419 0.1624 0.0922 0.8803 0.2330 0.1207 0.0693 2420 0.0372 0.0394 0.1086 0.0511 0.0214 0.0204 2422 0.0598 0.0509 0.9172 0.2393 0.0083 0.0108 2423 0.0094 0.0213 0.2131 0.0861 0.0000 0.0004 2426 0.0005 0.0054 0.2399 0.0932 0.0087 0.0109 2429 0.2051 0.1030 0.4357 0.1439 0.1204 0.0693 2431 0.0469 0.0435 0.6202 0.1825 0.1842 0.0969 2432 0.0083 0.0209 0.7253 0.2027 0.1761 0.0942 2442 0.1478 0.0859 0.8064 0.2193 0.7849 0.2891 2443 0.0163 0.0285 0.0428 0.0285 0.0537 0.0374 2445 0.0450 0.0428 0.3723 0.1296 0.0526 0.0372 2448 0.5207 0.1970 0.5978 0.1787 0.0149 0.0161 2449 0.0055 0.0186 0.0219 0.0186 0.0002 0.0014 2474 0.1808 0.0961 0.1395 0.0619 0.0590 0.0404 2480 0.0045 0.0169 0.0332 0.0243 0.0005 0.0022 2481 0.0371 0.0394 0.0107 0.0120 0.0001 0.0008 2488 0.0029 0.0150 0.0703 0.0385 0.0000 0.0004 2491 0.0167 0.0286 0.0024 0.0055 0.0004 0.0020 2493 0.0060 0.0195 0.0026 0.0055 0.0002 0.0014 2498 0.0885 0.0623 0.0306 0.0232 0.0016 0.0038 2499 0.0104 0.0222 0.0130 0.0134 0.0044 0.0072 2513 0.0028 0.0145 0.1401 0.0619 0.0019 0.0042 2514 0.0106 0.0222 0.0012 0.0040 0.0001 0.0008 2517 0.0194 0.0310 0.0676 0.0378 0.0012 0.0034 2518 0.0003 0.0041 0.0025 0.0055 0.0008 0.0027 2520 0.1306 0.0780 0.4579 0.1482 0.2646 0.1277 2522 0.8132 0.2655 0.4760 0.1514 0.3804 0.1653 2523 0.1104 0.0713 0.0936 0.0457 0.0270 0.0234 2525 0.2281 0.1096 0.2717 0.1028 0.7022 0.2668 2543 0.3249 0.1413 0.5644 0.1712 0.5335 0.2182 2547 0.0437 0.0428 0.4813 0.1520 0.8825 0.3131 2549 0.7965 0.2620 0.4758 0.1514 0.2817 0.1339 2561 0.0273 0.0345 0.0440 0.0290 0.0412 0.0313 2563 0.1660 0.0923 0.0064 0.0086 0.0079 0.0107 2564 0.0206 0.0316 0.0029 0.0060 0.0013 0.0034 2565 0.7399 0.2482 0.1970 0.0818 0.8012 0.2923 2566 0.0719 0.0548 0.0466 0.0302 0.1348 0.0754 2567 0.6504 0.2261 0.0375 0.0264 0.0429 0.0321 2568 0.0958 0.0642 0.0030 0.0060 0.0009 0.0027 2569 0.0424 0.0418 0.0188 0.0171 0.1251 0.0708 2570 0.0657 0.0521 0.0014 0.0042 0.0126 0.0147 2575 0.5810 0.2132 0.2394 0.0932 0.7882 0.2892 2577 0.6112 0.2184 0.0852 0.0430 0.2963 0.1388 2579 0.1690 0.0924 0.0378 0.0264 0.3443 0.1542 2580 0.0682 0.0531 0.0007 0.0032 0.0045 0.0072 2581 0.5201 0.1970 0.0367 0.0262 0.1406 0.0782 2583 0.1795 0.0960 0.0045 0.0069 0.0048 0.0076 2588 0.2229 0.1081 0.0267 0.0217 0.0728 0.0481 2591 0.3315 0.1431 0.0211 0.0183 0.0040 0.0066 2592 0.0085 0.0209 0.0161 0.0152 0.4130 0.1744 2594 0.1249 0.0764 0.0578 0.0343 0.0357 0.0288 2610 0.0764 0.0567 0.0013 0.0040 0.0039 0.0065 2624 0.3112 0.1367 0.0280 0.0222 0.0088 0.0110 2625 0.1087 0.0705 0.0212 0.0183 0.0052 0.0080 2649 0.3812 0.1563 0.0407 0.0278 0.8738 0.3112 2652 0.2180 0.1073 0.0672 0.0377 0.0066 0.0093 2669 0.6185 0.2196 0.0772 0.0405 0.8669 0.3093 3453 0.0242 0.0336 0.0542 0.0334 0.0031 0.0056 3454 0.0444 0.0428 0.0494 0.0312 0.0012 0.0033 3455 0.0019 0.0111 0.0012 0.0040 0.0000 0.0002 3456 0.1786 0.0958 0.0475 0.0305 0.0014 0.0034 3458 0.0003 0.0041 0.0613 0.0352 0.0000 0.0002 3461 0.1908 0.0992 0.0125 0.0132 0.0263 0.0231 3462 0.3962 0.1610 0.2241 0.0887 0.0066 0.0093 3464 0.3465 0.1470 0.0005 0.0026 0.0306 0.0253 3466 0.0265 0.0342 0.0021 0.0053 0.0006 0.0022 3471 0.9736 0.3020 0.0529 0.0331 0.6629 0.2558 3472 0.9410 0.2935 0.8073 0.2193 0.3894 0.1685 3473 0.3425 0.1467 0.1141 0.0526 0.4976 0.2061 3474 0.1635 0.0923 0.0402 0.0275 0.6912 0.2636 3478 0.0289 0.0345 0.0589 0.0345 0.0095 0.0116 3479 0.1493 0.0865 0.1882 0.0790 0.5662 0.2280 3480 0.5984 0.2156 0.8916 0.2354 0.9427 0.3261 3485 0.0114 0.0232 0.0012 0.0040 0.0008 0.0027 3495 0.0979 0.0651 0.0041 0.0067 0.0034 0.0059 3498 0.5917 0.2140 0.1733 0.0737 0.2553 0.1240 3505 0.0114 0.0232 0.0556 0.0337 0.0013 0.0034 3507 0.0797 0.0586 0.0106 0.0120 0.0228 0.0214 3508 0.0021 0.0118 0.0537 0.0332 0.0009 0.0028 3513 0.2646 0.1217 0.6812 0.1968 0.3490 0.1549 3514 0.3741 0.1548 0.4891 0.1535 0.6556 0.2546 3516 0.0065 0.0199 0.0014 0.0042 0.0012 0.0033 3522 0.1280 0.0775 0.0035 0.0064 0.0298 0.0247 3528 0.2914 0.1299 0.0835 0.0427 0.0161 0.0169 3530 0.6132 0.2186 0.7112 0.2003 0.4071 0.1735 3533 0.2981 0.1322 0.4774 0.1515 0.0028 0.0055 3534 0.8764 0.2804 0.6160 0.1816 0.0061 0.0089 3535 0.9155 0.2896 0.0937 0.0457 0.6611 0.2557 3537 0.0338 0.0377 0.0151 0.0149 0.0419 0.0316 3541 0.1127 0.0719 0.6886 0.1974 0.0873 0.0549 3542 0.7662 0.2549 0.9583 0.2475 0.2509 0.1226 3547 0.0007 0.0065 0.0077 0.0096 0.0000 0.0002 3548 0.0010 0.0080 0.0613 0.0352 0.0001 0.0006 3549 0.7416 0.2482 0.0088 0.0105 0.0532 0.0374 3550 0.5658 0.2094 0.0190 0.0171 0.0142 0.0156 3552 0.4037 0.1629 0.4328 0.1436 0.0426 0.0320 3563 0.4968 0.1904 0.1138 0.0526 0.2681 0.1291 3565 0.1300 0.0780 0.0420 0.0281 0.0635 0.0427 3566 0.1118 0.0717 0.0036 0.0064 0.0015 0.0037 3567 0.0657 0.0521 0.0038 0.0065 0.0053 0.0080 3568 0.0569 0.0489 0.0192 0.0172 0.7120 0.2700 3572 0.0334 0.0377 0.5222 0.1604 0.0886 0.0554 3573 0.2473 0.1167 0.8240 0.2228 0.5571 0.2259 3576 0.0102 0.0222 0.0288 0.0222 0.0221 0.0210 3577 0.0001 0.0026 0.0033 0.0064 0.0000 0.0002 3578 0.2476 0.1167 0.3862 0.1326 0.9624 0.3309 3579 0.0043 0.0169 0.0034 0.0064 0.0011 0.0033 3580 0.0954 0.0642 0.0111 0.0123 0.0250 0.0227 3585 0.0252 0.0338 0.5135 0.1588 0.2787 0.1328 3587 0.0000 0.0001 0.0001 0.0007 0.0000 0.0000 3588 0.0078 0.0209 0.0005 0.0026 0.0003 0.0017 3589 0.0059 0.0193 0.0008 0.0033 0.0013 0.0034 3598 0.0039 0.0164 0.0367 0.0262 0.0003 0.0014 3599 0.6955 0.2387 0.2867 0.1057 0.7785 0.2875 3602 0.0654 0.0521 0.7969 0.2177 0.3002 0.1400 3603 0.0080 0.0209 0.0315 0.0235 0.0411 0.0313 3607 0.0470 0.0435 0.5630 0.1712 0.0297 0.0247 3608 0.0223 0.0321 0.8988 0.2365 0.0510 0.0366 3624 0.0054 0.0186 0.0067 0.0089 0.0055 0.0083 3627 0.6088 0.2180 0.0478 0.0305 0.0782 0.0511 3628 0.1849 0.0975 0.1812 0.0765 0.3182 0.1462 3636 0.0064 0.0199 0.0597 0.0347 0.0392 0.0305 3641 0.4532 0.1780 0.0025 0.0055 0.0005 0.0022 3643 0.0003 0.0041 0.3915 0.1336 0.5707 0.2290 3644 0.0040 0.0164 0.7307 0.2038 0.9146 0.3192 3648 0.0849 0.0610 0.3833 0.1320 0.0461 0.0341 3649 0.0424 0.0418 0.0007 0.0030 0.0005 0.0022 3650 0.2000 0.1020 0.3075 0.1118 0.0965 0.0587 3651 0.6854 0.2376 0.0044 0.0069 0.0002 0.0014 3653 0.0092 0.0213 0.8277 0.2228 0.0163 0.0170 3654 0.0761 0.0567 0.0481 0.0305 0.0002 0.0011 3656 0.3493 0.1475 0.7140 0.2004 0.4046 0.1732 3658 0.0368 0.0394 0.3339 0.1189 0.0258 0.0229 3662 0.9285 0.2917 0.3212 0.1153 0.8509 0.3064 3663 0.6881 0.2376 0.0771 0.0405 0.4082 0.1735 3664 0.8371 0.2708 0.0375 0.0264 0.6870 0.2626 3667 0.9126 0.2896 0.1669 0.0717 0.9067 0.3176 3668 0.0261 0.0342 0.0056 0.0078 0.0215 0.0204 3670 0.4144 0.1653 0.3356 0.1193 0.7271 0.2752 3694 0.4864 0.1893 0.0004 0.0025 0.0017 0.0038 3698 0.5218 0.1970 0.7344 0.2041 0.2026 0.1032 3714 0.0031 0.0157 0.6019 0.1792 0.0027 0.0053 3715 0.0103 0.0222 0.3680 0.1286 0.0731 0.0482 3718 0.2237 0.1081 0.9102 0.2379 0.5205 0.2144 3727 0.0000 0.0008 0.0001 0.0006 0.0000 0.0000 3728 0.3102 0.1366 0.1275 0.0577 0.1000 0.0603 3751 0.1212 0.0752 0.7036 0.1994 0.0008 0.0027 3754 0.2859 0.1285 0.0837 0.0427 0.0032 0.0056 3757 0.0000 0.0008 0.0000 0.0000 0.0000 0.0000 3771 0.6871 0.2376 0.8631 0.2297 0.8469 0.3061 3978 0.5977 0.2156 0.1399 0.0619 0.7387 0.2790 4000 0.7347 0.2473 0.0932 0.0457 0.2557 0.1240 4079 0.8714 0.2798 0.1472 0.0646 0.0621 0.0420 4108 0.0336 0.0377 0.0000 0.0001 0.0000 0.0004 4131 0.7310 0.2468 0.9343 0.2433 0.6398 0.2520 4144 0.6993 0.2391 0.0197 0.0175 0.1272 0.0716 4221 0.0223 0.0321 0.0000 0.0000 0.0000 0.0003 4246 0.0340 0.0377 0.0068 0.0090 0.6059 0.2406 4274 0.8733 0.2799 0.0036 0.0064 0.0003 0.0016 4363 0.8887 0.2838 0.0681 0.0379 0.2316 0.1149 4756 0.4899 0.1898 0.0583 0.0344 0.0979 0.0594 4832 0.2738 0.1246 0.2556 0.0980 0.4107 0.1738 4835 0.7093 0.2411 0.0004 0.0025 0.0027 0.0053 4899 0.1277 0.0775 0.5813 0.1745 0.0416 0.0315 4926 0.8925 0.2845 0.8925 0.2354 0.0070 0.0097 4930 0.0000 0.0001 0.2618 0.0998 0.0828 0.0532 4971 0.1580 0.0910 0.5798 0.1745 0.1922 0.1003

TABLE 4E Sp/ABX GF/ABX GF/Sp PSO p-value q-value p-value q-value p-value q-value 1 0.8917 0.5563 0.1267 0.3091 0.1609 0.5562 2 0.5072 0.4333 0.2155 0.3855 0.0648 0.4168 5 0.3900 0.3727 0.0802 0.2676 0.3465 0.7016 6 0.0506 0.1713 0.0562 0.2376 0.9585 0.8691 9 0.0641 0.1838 0.7898 0.6001 0.1067 0.5131 10 0.8368 0.5346 0.2106 0.3803 0.1487 0.5562 14 0.3008 0.3317 0.1547 0.3609 0.0194 0.2317 16 0.8637 0.5475 0.2699 0.4251 0.2055 0.5984 17 0.5051 0.4333 0.0564 0.2376 0.1932 0.5864 18 0.5041 0.4333 0.4624 0.5230 0.9457 0.8691 21 0.3804 0.3666 0.3223 0.4661 0.9073 0.8681 28 0.8984 0.5563 0.7342 0.5835 0.6409 0.8251 30 0.1043 0.2278 0.0667 0.2508 0.8144 0.8519 34 0.9011 0.5563 0.8090 0.6041 0.9064 0.8681 35 0.2546 0.2999 0.6489 0.5695 0.4855 0.7453 38 0.9840 0.5782 0.9418 0.6284 0.9258 0.8691 39 0.7121 0.4942 0.4316 0.5036 0.6730 0.8315 40 0.1827 0.2616 0.9116 0.6254 0.2193 0.6064 41 0.5689 0.4508 0.6138 0.5695 0.9476 0.8691 42 0.0500 0.1713 0.5935 0.5688 0.0161 0.2066 43 0.0000 0.0007 0.1850 0.3746 0.0000 0.0001 44 0.8981 0.5563 0.8933 0.6233 0.9951 0.8795 45 0.1288 0.2311 0.7327 0.5835 0.0679 0.4298 47 0.0001 0.0019 0.9421 0.6284 0.0000 0.0026 50 0.3096 0.3368 0.2838 0.4431 0.9536 0.8691 51 0.0359 0.1496 0.0114 0.1024 0.0001 0.0026 52 0.6346 0.4691 0.4478 0.5190 0.2233 0.6097 53 0.5166 0.4340 0.3668 0.4776 0.1289 0.5309 54 0.5193 0.4340 0.6166 0.5695 0.2579 0.6316 55 0.7881 0.5248 0.2108 0.3803 0.3198 0.6817 56 0.0863 0.2155 0.0374 0.2039 0.6752 0.8321 59 0.0394 0.1566 0.0119 0.1042 0.5790 0.7830 65 0.1339 0.2311 0.1067 0.2848 0.9002 0.8681 66 0.0024 0.0325 0.0010 0.0306 0.7145 0.8380 67 0.2056 0.2747 0.0711 0.2551 0.5567 0.7770 68 0.0509 0.1713 0.7325 0.5835 0.0250 0.2775 74 0.0000 0.0000 0.9507 0.6291 0.0000 0.0000 75 0.0512 0.1713 0.2229 0.3875 0.4242 0.7283 76 0.1850 0.2618 0.3984 0.4995 0.0369 0.3243 77 0.7740 0.5212 0.6904 0.5790 0.9111 0.8681 80 0.0181 0.0987 0.8231 0.6041 0.0111 0.1537 82 0.2972 0.3287 0.9509 0.6291 0.3254 0.6900 83 0.2580 0.3016 0.7178 0.5835 0.4344 0.7283 84 0.4436 0.4017 0.6744 0.5775 0.7261 0.8380 92 0.8109 0.5274 0.4620 0.5230 0.3329 0.6976 93 0.0100 0.0718 0.5070 0.5413 0.0423 0.3453 99 0.0217 0.1120 0.2581 0.4219 0.0016 0.0466 100 0.1137 0.2311 0.9466 0.6291 0.1005 0.5102 101 0.2485 0.2999 0.6493 0.5695 0.4757 0.7453 102 0.1453 0.2422 0.1714 0.3733 0.9239 0.8691 103 0.4127 0.3838 0.4558 0.5230 0.9401 0.8691 105 0.3363 0.3473 0.7033 0.5824 0.5560 0.7770 107 0.7779 0.5212 0.6755 0.5775 0.4855 0.7453 113 0.1309 0.2311 0.1766 0.3736 0.0075 0.1289 114 0.7008 0.4913 0.1085 0.2848 0.2116 0.6023 117 0.4347 0.3960 0.3941 0.4991 0.1109 0.5146 119 0.0000 0.0000 0.0059 0.0770 0.0000 0.0000 120 0.3416 0.3473 0.6244 0.5695 0.1567 0.5562 121 0.2413 0.2964 0.8079 0.6041 0.1615 0.5562 122 0.4630 0.4136 0.8773 0.6181 0.5606 0.7770 126 0.3134 0.3388 0.9179 0.6254 0.2684 0.6409 137 0.4537 0.4086 0.2240 0.3875 0.0571 0.4034 138 0.6950 0.4913 0.0519 0.2360 0.0229 0.2599 150 0.0678 0.1925 0.6914 0.5790 0.0301 0.2992 154 0.1614 0.2443 1.0000 0.6499 0.1614 0.5562 180 0.8101 0.5274 0.8403 0.6048 0.6592 0.8268 181 0.7774 0.5212 0.4219 0.5036 0.2816 0.6480 191 0.1771 0.2595 0.2268 0.3879 0.8807 0.8620 210 0.7774 0.5212 0.5512 0.5606 0.7526 0.8380 217 0.2425 0.2964 0.6882 0.5790 0.4347 0.7283 221 0.6866 0.4905 0.4818 0.5299 0.2734 0.6411 227 0.0570 0.1757 0.0395 0.2100 0.8559 0.8610 241 0.2015 0.2715 0.6268 0.5695 0.4182 0.7283 254 0.0078 0.0610 0.0000 0.0068 0.0000 0.0000 261 0.6739 0.4870 0.8377 0.6041 0.5328 0.7770 263 0.4187 0.3872 0.3589 0.4766 0.9109 0.8681 265 0.8984 0.5563 0.5609 0.5622 0.4794 0.7453 273 0.7334 0.5011 0.1088 0.2848 0.1975 0.5917 280 0.0391 0.1566 0.3214 0.4661 0.2478 0.6228 296 0.1886 0.2645 0.8972 0.6233 0.2328 0.6153 299 0.2279 0.2893 0.9055 0.6254 0.1876 0.5850 300 0.2365 0.2953 0.5503 0.5606 0.5469 0.7770 301 0.0108 0.0724 0.1225 0.3029 0.2442 0.6224 303 0.7027 0.4913 0.6393 0.5695 0.9302 0.8691 306 0.9759 0.5766 0.7557 0.5925 0.7787 0.8418 311 0.0015 0.0272 0.0376 0.2039 0.1616 0.5562 312 0.5547 0.4482 0.1987 0.3759 0.4748 0.7453 318 0.8247 0.5300 0.6009 0.5693 0.7619 0.8380 321 0.0378 0.1538 0.1191 0.3026 0.5578 0.7770 323 0.6526 0.4749 0.9420 0.6284 0.7055 0.8380 326 0.9918 0.5795 0.3433 0.4761 0.3484 0.7016 328 0.6245 0.4679 0.0968 0.2845 0.2275 0.6153 332 0.2433 0.2964 0.8078 0.6041 0.3507 0.7016 333 0.0003 0.0076 0.0134 0.1146 0.1008 0.5102 334 0.4757 0.4222 0.7546 0.5925 0.6862 0.8380 337 0.0047 0.0517 0.0052 0.0717 0.9615 0.8691 342 0.1942 0.2690 0.6750 0.5775 0.0922 0.5044 343 0.4133 0.3838 0.3604 0.4766 0.9210 0.8691 349 0.1327 0.2311 0.1766 0.3736 0.8693 0.8610 351 0.0404 0.1585 0.0266 0.1631 0.8416 0.8610 353 0.1294 0.2311 0.4200 0.5036 0.4572 0.7415 362 0.8135 0.5274 0.3491 0.4761 0.2450 0.6224 363 0.0229 0.1162 0.7891 0.6001 0.0128 0.1680 364 0.4589 0.4111 0.5792 0.5622 0.2021 0.5984 365 0.1078 0.2298 0.0720 0.2551 0.8312 0.8558 367 0.5179 0.4340 0.1322 0.3205 0.3730 0.7152 369 0.0000 0.0004 0.5749 0.5622 0.0000 0.0009 371 0.9686 0.5743 0.4009 0.4995 0.4228 0.7283 372 0.6384 0.4708 0.7492 0.5914 0.4324 0.7283 373 0.1835 0.2616 0.5921 0.5688 0.4146 0.7283 375 0.6029 0.4610 0.2477 0.4074 0.5154 0.7653 379 0.9052 0.5567 0.8506 0.6098 0.7587 0.8380 380 0.8005 0.5274 0.4689 0.5230 0.3318 0.6976 381 0.5163 0.4340 0.3312 0.4749 0.1132 0.5146 382 0.3435 0.3473 0.0004 0.0215 0.0000 0.0026 383 0.6292 0.4691 0.6280 0.5695 0.3376 0.6981 386 0.0570 0.1757 0.6894 0.5790 0.1221 0.5308 387 0.7305 0.5006 0.5623 0.5622 0.8128 0.8519 392 0.1278 0.2311 0.0025 0.0487 0.0001 0.0026 397 0.1012 0.2257 0.6055 0.5695 0.2466 0.6228 399 0.1239 0.2311 0.8032 0.6041 0.1910 0.5863 405 0.9950 0.5795 0.6737 0.5775 0.6691 0.8314 410 0.6520 0.4749 0.1169 0.2991 0.2514 0.6228 411 0.2790 0.3161 0.8906 0.6233 0.3420 0.7014 412 0.4963 0.4305 0.2211 0.3875 0.5750 0.7827 418 0.3217 0.3454 0.4188 0.5036 0.8508 0.8610 421 0.5870 0.4576 0.7796 0.5988 0.7911 0.8447 426 0.6146 0.4647 0.8137 0.6041 0.7879 0.8447 429 0.2543 0.2999 0.5647 0.5622 0.5630 0.7782 430 0.0292 0.1325 0.0875 0.2729 0.5868 0.7858 436 0.0058 0.0536 0.0112 0.1024 0.7702 0.8418 438 0.9896 0.5795 0.9261 0.6282 0.9364 0.8691 439 0.2011 0.2715 0.8897 0.6233 0.2513 0.6228 442 0.1372 0.2318 0.0766 0.2639 0.7533 0.8380 443 0.6099 0.4633 0.5663 0.5622 0.9489 0.8691 445 0.1297 0.2311 0.1574 0.3616 0.9125 0.8681 446 0.4198 0.3872 0.6980 0.5811 0.2376 0.6213 447 0.0065 0.0556 0.0007 0.0251 0.3406 0.7013 451 0.3489 0.3474 0.7264 0.5835 0.2037 0.5984 454 0.4529 0.4086 0.3514 0.4761 0.1009 0.5102 456 0.1567 0.2443 0.4118 0.5036 0.0317 0.2992 459 0.0066 0.0556 0.0718 0.2551 0.2715 0.6409 460 0.7922 0.5254 0.2051 0.3759 0.1305 0.5309 461 0.3841 0.3681 0.5082 0.5413 0.8310 0.8558 465 0.0773 0.2022 0.3118 0.4633 0.4192 0.7283 467 0.1101 0.2298 0.0435 0.2196 0.6344 0.8251 468 0.2810 0.3162 0.0624 0.2508 0.3968 0.7283 469 0.0594 0.1764 0.3463 0.4761 0.3133 0.6797 471 0.9604 0.5715 0.1405 0.3366 0.1532 0.5562 472 0.5430 0.4464 0.7291 0.5835 0.7917 0.8447 473 0.2141 0.2800 0.0205 0.1413 0.2321 0.6153 474 0.1243 0.2311 0.1644 0.3649 0.8736 0.8620 475 0.1726 0.2541 0.1588 0.3616 0.9610 0.8691 476 0.3067 0.3362 0.7248 0.5835 0.4969 0.7557 477 0.9531 0.5691 0.7816 0.5988 0.8270 0.8551 478 0.0872 0.2155 0.1477 0.3488 0.7731 0.8418 479 0.1962 0.2703 0.7802 0.5988 0.1209 0.5307 480 0.9258 0.5621 0.8309 0.6041 0.7593 0.8380 756 0.1600 0.2443 0.5439 0.5598 0.4098 0.7283 929 0.0303 0.1333 1.0000 0.6499 0.0303 0.2992 955 0.0930 0.2167 0.0001 0.0117 0.0000 0.0002 958 0.5888 0.4576 0.0053 0.0717 0.0178 0.2172 959 0.1041 0.2278 0.1597 0.3616 0.8112 0.8519 965 0.0316 0.1372 0.0461 0.2206 0.8557 0.8610 967 0.5598 0.4491 0.6986 0.5811 0.3360 0.6976 972 0.2604 0.3026 0.1420 0.3376 0.7150 0.8380 973 0.3242 0.3458 0.0951 0.2834 0.4671 0.7420 974 0.5643 0.4500 0.1966 0.3759 0.4621 0.7415 975 0.3391 0.3473 0.2258 0.3879 0.7897 0.8447 978 0.1822 0.2616 0.0926 0.2797 0.7048 0.8380 981 0.0036 0.0444 0.1025 0.2848 0.1295 0.5309 982 0.0918 0.2167 0.0954 0.2834 0.9838 0.8791 983 0.8477 0.5385 0.7611 0.5929 0.6205 0.8150 984 0.9778 0.5766 0.9290 0.6282 0.9069 0.8681 986 0.1900 0.2653 0.3179 0.4661 0.7430 0.8380 993 0.0640 0.1838 0.5981 0.5693 0.1695 0.5655 994 0.5374 0.4428 0.0337 0.1949 0.1141 0.5146 995 0.6207 0.4661 0.7094 0.5835 0.3890 0.7252 997 0.8871 0.5563 0.6314 0.5695 0.5352 0.7770 1018 0.0004 0.0121 0.0111 0.1024 0.1772 0.5832 1020 0.3512 0.3477 0.4874 0.5345 0.8075 0.8519 1023 0.0592 0.1764 0.2002 0.3759 0.5070 0.7596 1024 0.2063 0.2747 0.4318 0.5036 0.6198 0.8150 1028 0.2556 0.2999 0.8764 0.6181 0.1992 0.5933 1030 0.1493 0.2443 0.6480 0.5695 0.3124 0.6797 1033 0.4107 0.3838 0.4982 0.5368 0.8820 0.8620 1073 0.1032 0.2278 0.0201 0.1413 0.4227 0.7283 1078 0.1526 0.2443 0.6473 0.5695 0.3188 0.6817 1079 0.6450 0.4735 0.1106 0.2871 0.2436 0.6224 1080 0.0941 0.2167 0.0973 0.2845 0.9859 0.8794 1090 0.2351 0.2947 0.1609 0.3616 0.8190 0.8519 1099 0.8154 0.5274 0.9431 0.6284 0.7606 0.8380 1104 0.3839 0.3681 0.3514 0.4761 0.9497 0.8691 1108 0.5927 0.4576 0.1820 0.3746 0.4113 0.7283 1109 0.0585 0.1764 0.8297 0.6041 0.0889 0.4997 1111 0.2407 0.2964 0.1232 0.3029 0.6933 0.8380 1115 0.1584 0.2443 0.4610 0.5230 0.4836 0.7453 1116 0.7075 0.4931 0.4940 0.5353 0.7552 0.8380 1123 0.1154 0.2311 0.1488 0.3493 0.8866 0.8632 1127 0.4971 0.4305 0.5922 0.5688 0.8843 0.8626 1131 0.8069 0.5274 0.5793 0.5622 0.4268 0.7283 1132 0.0729 0.2002 0.0373 0.2039 0.7397 0.8380 1138 0.6885 0.4905 0.4302 0.5036 0.2396 0.6213 1149 0.6889 0.4905 0.6836 0.5790 0.9942 0.8795 1150 0.8072 0.5274 0.6913 0.5790 0.8779 0.8620 1152 0.9386 0.5646 0.5432 0.5598 0.4941 0.7538 1157 0.5546 0.4482 0.3481 0.4761 0.7227 0.8380 1163 0.8336 0.5336 0.5247 0.5509 0.6685 0.8314 1165 0.2018 0.2715 0.6608 0.5743 0.3923 0.7252 1166 0.6943 0.4913 0.5138 0.5441 0.3003 0.6708 1167 0.8192 0.5274 0.9094 0.6254 0.9085 0.8681 1169 0.1645 0.2464 0.7169 0.5835 0.0852 0.4957 1179 0.5044 0.4333 0.4052 0.5016 0.8663 0.8610 1181 0.1688 0.2495 0.1840 0.3746 0.9593 0.8691 1183 0.7897 0.5248 0.0049 0.0717 0.0026 0.0693 1201 0.4051 0.3805 0.8535 0.6107 0.5146 0.7653 1203 0.0926 0.2167 0.8176 0.6041 0.1411 0.5562 1204 0.3659 0.3590 0.9672 0.6353 0.3452 0.7016 1205 0.6329 0.4691 0.1569 0.3616 0.0646 0.4168 1217 0.1501 0.2443 0.2233 0.3875 0.8128 0.8519 1218 0.1080 0.2298 0.0339 0.1949 0.5582 0.7770 1223 0.2420 0.2964 0.5336 0.5566 0.5736 0.7827 1225 0.0787 0.2041 0.0797 0.2676 0.9945 0.8795 1236 0.1101 0.2298 0.5744 0.5622 0.2840 0.6489 1239 0.6055 0.4610 0.5774 0.5622 0.9673 0.8695 1244 0.1255 0.2311 0.3290 0.4736 0.5563 0.7770 1247 0.3954 0.3754 0.1727 0.3733 0.5914 0.7891 1253 0.1372 0.2318 0.6101 0.5695 0.3151 0.6797 1258 0.0050 0.0517 0.0012 0.0339 0.5403 0.7770 1259 0.0444 0.1640 0.0234 0.1513 0.7605 0.8380 1260 0.1095 0.2298 0.0511 0.2355 0.6935 0.8380 1264 0.0974 0.2214 0.0499 0.2355 0.7313 0.8380 1265 0.0330 0.1393 0.1052 0.2848 0.5594 0.7770 1267 0.3399 0.3473 0.1785 0.3736 0.6814 0.8357 1269 0.0297 0.1325 0.0655 0.2508 0.6994 0.8380 1270 0.1142 0.2311 0.0842 0.2676 0.8706 0.8610 1271 0.3725 0.3622 0.2997 0.4575 0.8804 0.8620 1272 0.3467 0.3473 0.3642 0.4776 0.9722 0.8705 1273 0.2251 0.2893 0.5853 0.5661 0.4938 0.7538 1274 0.1353 0.2318 0.2172 0.3866 0.7808 0.8418 1276 0.0703 0.1968 0.0760 0.2639 0.9679 0.8695 1277 0.2702 0.3106 0.3804 0.4867 0.8150 0.8519 1351 0.0246 0.1231 0.2014 0.3759 0.2807 0.6480 1355 0.8160 0.5274 0.6748 0.5775 0.8512 0.8610 1359 0.8479 0.5385 0.7363 0.5835 0.5979 0.7958 1360 0.7865 0.5248 0.1928 0.3759 0.2960 0.6642 1363 0.9411 0.5646 0.3941 0.4991 0.3555 0.7016 1372 0.0768 0.2022 0.7786 0.5988 0.1297 0.5309 1430 0.6811 0.4891 0.2009 0.3759 0.3760 0.7182 1446 0.0908 0.2167 0.0161 0.1236 0.4043 0.7283 1449 0.4880 0.4290 0.2339 0.3921 0.6081 0.8054 1452 0.0057 0.0536 0.0207 0.1413 0.5657 0.7798 1480 0.0876 0.2155 0.0158 0.1236 0.4103 0.7283 1482 0.8932 0.5563 0.2034 0.3759 0.1623 0.5562 1483 0.5727 0.4527 0.9634 0.6339 0.5422 0.7770 1485 0.0448 0.1640 0.0022 0.0478 0.1862 0.5850 1487 0.3523 0.3477 0.5522 0.5606 0.7317 0.8380 1488 0.0481 0.1705 0.0145 0.1190 0.5732 0.7827 1495 0.0265 0.1275 0.0006 0.0251 0.1077 0.5131 1496 0.0027 0.0354 0.0032 0.0542 0.9359 0.8691 1498 0.0049 0.0517 0.0313 0.1861 0.4069 0.7283 1499 0.0012 0.0250 0.0002 0.0150 0.4594 0.7415 1500 0.0120 0.0774 0.1777 0.3736 0.1868 0.5850 1501 0.0060 0.0536 0.0000 0.0068 0.0398 0.3434 1503 0.1279 0.2311 0.0034 0.0550 0.0992 0.5102 1504 0.0042 0.0494 0.0007 0.0251 0.4521 0.7415 1506 0.0071 0.0583 0.0002 0.0150 0.1233 0.5308 1517 0.0021 0.0325 0.3098 0.4633 0.0216 0.2516 1518 0.2135 0.2800 0.1219 0.3029 0.7443 0.8380 1519 0.1253 0.2311 0.0672 0.2508 0.7404 0.8380 1521 0.9421 0.5646 0.7271 0.5835 0.7821 0.8418 1522 0.0752 0.2002 0.0032 0.0542 0.1574 0.5562 1523 0.0186 0.0987 0.0112 0.1024 0.8197 0.8519 1524 0.0012 0.0250 0.0541 0.2376 0.1008 0.5102 1527 0.6329 0.4691 0.7653 0.5937 0.8571 0.8610 1528 0.9839 0.5782 0.3664 0.4776 0.3561 0.7016 1529 0.9418 0.5646 0.9150 0.6254 0.8574 0.8610 1536 0.2549 0.2999 0.0457 0.2206 0.3492 0.7016 1537 0.3395 0.3473 0.8190 0.6041 0.4640 0.7418 1539 0.6187 0.4657 0.0239 0.1518 0.0079 0.1319 1541 0.3704 0.3613 0.6673 0.5775 0.6364 0.8251 1542 0.3291 0.3473 0.3550 0.4766 0.9579 0.8691 1547 0.5510 0.4474 0.0349 0.1980 0.0095 0.1438 1565 0.0995 0.2247 0.1886 0.3753 0.7181 0.8380 1567 0.0514 0.1713 0.1082 0.2848 0.7006 0.8380 1582 0.6160 0.4647 0.9628 0.6339 0.6489 0.8251 1711 0.2539 0.2999 0.5787 0.5622 0.5486 0.7770 1718 0.5919 0.4576 0.1954 0.3759 0.4361 0.7283 1721 0.1512 0.2443 0.6197 0.5695 0.3348 0.6976 1731 0.9262 0.5621 0.9144 0.6254 0.9881 0.8795 1732 0.4243 0.3886 0.4206 0.5036 0.9947 0.8795 1744 0.5087 0.4333 0.5348 0.5566 0.9676 0.8695 1753 0.2935 0.3257 0.5427 0.5598 0.1050 0.5131 1754 0.0014 0.0272 0.4012 0.4995 0.0100 0.1466 1782 0.0594 0.1764 0.1090 0.2848 0.7514 0.8380 1783 0.5924 0.4576 0.7553 0.5925 0.3999 0.7283 1784 0.0174 0.0987 0.2076 0.3788 0.2118 0.6023 1786 0.0000 0.0007 0.0004 0.0215 0.1142 0.5146 1788 0.2512 0.2999 0.7611 0.5929 0.1518 0.5562 1789 0.2869 0.3217 0.7359 0.5835 0.4608 0.7415 1790 0.0089 0.0665 0.1755 0.3736 0.1507 0.5562 1791 0.8874 0.5563 0.3204 0.4661 0.2587 0.6316 1792 0.5911 0.4576 0.0275 0.1661 0.0819 0.4886 1793 0.0000 0.0008 0.0089 0.0919 0.0000 0.0000 1811 0.2616 0.3026 0.8182 0.6041 0.1804 0.5850 1815 0.6708 0.4860 0.8005 0.6041 0.8626 0.8610 1827 0.2900 0.3230 0.6911 0.5790 0.5020 0.7580 1828 0.5922 0.4576 0.5475 0.5606 0.2612 0.6345 1829 0.0363 0.1496 0.3586 0.4766 0.2069 0.5984 1831 0.3354 0.3473 0.8571 0.6114 0.4304 0.7283 1834 0.1364 0.2318 0.9269 0.6282 0.1602 0.5562 1837 0.2408 0.2964 0.7994 0.6041 0.1581 0.5562 1841 0.0260 0.1275 0.4799 0.5295 0.1075 0.5131 1845 0.2742 0.3117 0.7228 0.5835 0.4534 0.7415 1851 0.7175 0.4958 0.1004 0.2848 0.1904 0.5863 1866 0.4976 0.4305 0.2195 0.3875 0.0643 0.4168 1867 0.5923 0.4576 0.3013 0.4575 0.1241 0.5308 1870 0.1911 0.2658 0.5162 0.5450 0.0577 0.4034 1878 0.5989 0.4592 0.3385 0.4761 0.1454 0.5562 1884 0.5446 0.4466 0.0735 0.2580 0.2178 0.6063 1889 0.4130 0.3838 0.0684 0.2522 0.2887 0.6507 1892 0.0105 0.0719 0.7495 0.5914 0.0051 0.1021 1902 0.2507 0.2999 0.0530 0.2376 0.3927 0.7252 1903 0.0020 0.0325 0.9348 0.6284 0.0024 0.0680 1907 0.1561 0.2443 0.8592 0.6114 0.2104 0.6023 1945 0.9674 0.5743 0.4211 0.5036 0.4446 0.7345 1948 0.2513 0.2999 0.7634 0.5935 0.1528 0.5562 1949 0.5600 0.4491 0.1070 0.2848 0.2865 0.6507 1950 0.0497 0.1713 0.2671 0.4245 0.3546 0.7016 1953 0.3117 0.3380 0.4666 0.5230 0.7705 0.8418 1955 0.3568 0.3511 0.0674 0.2508 0.3336 0.6976 1962 0.9114 0.5574 0.1815 0.3746 0.2181 0.6063 1970 0.4974 0.4305 0.1732 0.3733 0.4791 0.7453 1974 0.3480 0.3474 0.0505 0.2355 0.2760 0.6423 1975 0.1455 0.2422 0.2588 0.4219 0.7282 0.8380 1976 0.0216 0.1120 0.4143 0.5036 0.1128 0.5146 1979 0.1612 0.2443 0.3770 0.4841 0.5873 0.7858 1982 0.3398 0.3473 0.1021 0.2848 0.4705 0.7431 1985 0.0467 0.1693 0.5029 0.5402 0.1661 0.5616 2016 0.6755 0.4870 0.8330 0.6041 0.8348 0.8578 2021 0.9932 0.5795 0.6421 0.5695 0.6482 0.8251 2024 0.5150 0.4340 0.4712 0.5230 0.9437 0.8691 2034 0.1335 0.2311 0.9181 0.6254 0.1599 0.5562 2052 0.5201 0.4340 0.1615 0.3616 0.4337 0.7283 2067 0.6403 0.4711 0.3621 0.4766 0.6517 0.8251 2072 0.1572 0.2443 0.1039 0.2848 0.8171 0.8519 2105 0.1672 0.2483 0.7675 0.5941 0.2704 0.6409 2106 0.0634 0.1838 0.3699 0.4799 0.3070 0.6785 2107 0.1812 0.2616 0.5744 0.5622 0.4250 0.7283 2113 0.1153 0.2311 0.4629 0.5230 0.3799 0.7228 2115 0.1968 0.2703 0.8429 0.6055 0.2700 0.6409 2118 0.9029 0.5563 0.4954 0.5353 0.4230 0.7283 2136 0.7034 0.4913 0.4358 0.5066 0.6869 0.8380 2157 0.1619 0.2443 0.6697 0.5775 0.3201 0.6817 2164 0.3501 0.3476 0.6313 0.5695 0.1642 0.5589 2165 0.5508 0.4474 0.6280 0.5695 0.9098 0.8681 2166 0.2437 0.2964 0.6778 0.5782 0.4447 0.7345 2168 0.8103 0.5274 0.3486 0.4761 0.4818 0.7453 2179 0.1591 0.2443 0.2351 0.3921 0.8139 0.8519 2192 0.2607 0.3026 0.3177 0.4661 0.8957 0.8681 2193 0.0433 0.1640 0.1861 0.3748 0.4394 0.7305 2194 0.8745 0.5533 0.7578 0.5929 0.6416 0.8251 2199 0.2222 0.2893 0.0840 0.2676 0.5829 0.7858 2203 0.2282 0.2893 0.4013 0.4995 0.7039 0.8380 2206 0.7021 0.4913 0.6515 0.5695 0.4072 0.7283 2209 0.7281 0.5004 0.6080 0.5695 0.3927 0.7252 2211 0.3753 0.3628 0.7981 0.6041 0.5246 0.7740 2217 0.0942 0.2167 0.0646 0.2508 0.8445 0.8610 2310 0.1841 0.2616 0.1950 0.3759 0.9724 0.8705 2311 0.0447 0.1640 0.2344 0.3921 0.3707 0.7146 2312 0.5323 0.4420 0.2612 0.4221 0.0883 0.4997 2313 0.5651 0.4500 0.6088 0.5695 0.2823 0.6480 2314 0.1623 0.2443 0.3326 0.4750 0.6520 0.8251 2318 0.2100 0.2772 0.1137 0.2931 0.7236 0.8380 2327 0.2886 0.3225 0.3721 0.4811 0.8613 0.8610 2343 0.7723 0.5212 0.2678 0.4245 0.1673 0.5618 2345 0.2277 0.2893 0.0415 0.2154 0.3614 0.7037 2347 0.9766 0.5766 0.0877 0.2729 0.0927 0.5044 2348 0.1321 0.2311 0.2274 0.3879 0.7487 0.8380 2350 0.9394 0.5646 0.8270 0.6041 0.7685 0.8418 2351 0.0549 0.1757 0.1667 0.3679 0.5529 0.7770 2356 0.3408 0.3473 0.2658 0.4245 0.8675 0.8610 2357 0.5479 0.4474 0.8603 0.6114 0.6696 0.8314 2360 0.6340 0.4691 0.6009 0.5693 0.9622 0.8691 2361 0.4778 0.4222 0.7888 0.6001 0.6561 0.8251 2364 0.4396 0.3992 0.6469 0.5695 0.7497 0.8380 2371 0.1537 0.2443 0.8214 0.6041 0.2242 0.6097 2374 0.0871 0.2155 0.3760 0.4841 0.3822 0.7245 2375 0.7645 0.5185 0.8326 0.6041 0.9295 0.8691 2376 0.8157 0.5274 0.7779 0.5988 0.9608 0.8691 2379 0.9018 0.5563 0.5705 0.5622 0.6562 0.8251 2381 0.4894 0.4290 0.5093 0.5413 0.1840 0.5850 2383 0.1840 0.2616 0.3225 0.4661 0.7214 0.8380 2394 0.1243 0.2311 0.2913 0.4492 0.6084 0.8054 2395 0.0130 0.0808 0.6430 0.5695 0.0354 0.3175 2407 0.9444 0.5650 0.4218 0.5036 0.4623 0.7415 2411 0.8949 0.5563 0.6834 0.5790 0.7823 0.8418 2413 0.0095 0.0696 0.1895 0.3753 0.1468 0.5562 2416 0.2734 0.3117 0.4735 0.5240 0.6964 0.8380 2418 0.0128 0.0808 0.5694 0.5622 0.0435 0.3453 2419 0.1246 0.2311 0.0914 0.2784 0.8665 0.8610 2420 0.5868 0.4576 0.4232 0.5036 0.7935 0.8449 2422 0.0734 0.2002 0.0104 0.1024 0.3603 0.7037 2423 0.1282 0.2311 0.0007 0.0251 0.0270 0.2876 2426 0.0076 0.0609 0.1055 0.2848 0.2171 0.6063 2429 0.6126 0.4643 0.4182 0.5036 0.7580 0.8380 2431 0.1221 0.2311 0.3934 0.4991 0.4665 0.7420 2432 0.0181 0.0987 0.3080 0.4633 0.1423 0.5562 2442 0.2232 0.2893 0.9776 0.6396 0.2334 0.6153 2443 0.6514 0.4749 0.9108 0.6254 0.5737 0.7827 2445 0.2344 0.2947 0.2646 0.4245 0.9384 0.8691 2448 0.9076 0.5571 0.0461 0.2206 0.0582 0.4034 2449 0.5356 0.4425 0.0595 0.2435 0.1866 0.5850 2474 0.8804 0.5560 0.6482 0.5695 0.5452 0.7770 2480 0.3736 0.3623 0.0830 0.2676 0.3713 0.7146 2481 0.5681 0.4508 0.0567 0.2376 0.0170 0.2130 2488 0.1556 0.2443 0.0031 0.0542 0.0738 0.4614 2491 0.3989 0.3757 0.4702 0.5230 0.1257 0.5309 2493 0.7144 0.4948 0.3117 0.4633 0.1742 0.5773 2498 0.5980 0.4592 0.2048 0.3759 0.0796 0.4860 2499 0.9211 0.5621 0.6360 0.5695 0.7077 0.8380 2513 0.0751 0.2002 0.0559 0.2376 0.8802 0.8620 2514 0.3463 0.3473 0.3045 0.4599 0.0572 0.4034 2517 0.5486 0.4474 0.0832 0.2676 0.2391 0.6213 2518 0.3423 0.3473 0.6378 0.5695 0.6262 0.8182 2520 0.4220 0.3875 0.7001 0.5811 0.6725 0.8315 2522 0.3457 0.3473 0.8662 0.6127 0.2692 0.6409 2523 0.9294 0.5621 0.5386 0.5590 0.4826 0.7453 2525 0.9111 0.5574 0.4665 0.5230 0.4024 0.7283 2543 0.6767 0.4870 0.9625 0.6339 0.7112 0.8380 2547 0.1665 0.2483 0.5764 0.5622 0.0588 0.4034 2549 0.6467 0.4737 0.0817 0.2676 0.1866 0.5850 2561 0.8192 0.5274 0.9742 0.6388 0.8444 0.8610 2563 0.1241 0.2311 0.9291 0.6282 0.1453 0.5562 2564 0.3928 0.3742 0.7265 0.5835 0.2336 0.6153 2565 0.3303 0.3473 0.2933 0.4503 0.9358 0.8691 2566 0.8272 0.5305 0.5797 0.5622 0.7361 0.8380 2567 0.0922 0.2167 0.9478 0.6291 0.1042 0.5131 2568 0.1188 0.2311 0.5943 0.5688 0.0421 0.3453 2569 0.7019 0.4913 0.3509 0.4761 0.5772 0.7827 2570 0.0943 0.2167 0.3472 0.4761 0.4366 0.7283 2575 0.5220 0.4345 0.3582 0.4766 0.7757 0.8418 2577 0.2102 0.2772 0.4688 0.5230 0.5844 0.7858 2579 0.4349 0.3960 0.2238 0.3875 0.6515 0.8251 2580 0.0542 0.1757 0.4499 0.5197 0.2169 0.6063 2581 0.1288 0.2311 0.4892 0.5349 0.3895 0.7252 2583 0.0863 0.2155 0.9790 0.6396 0.0907 0.5044 2588 0.2704 0.3106 0.6243 0.5695 0.5320 0.7770 2591 0.1471 0.2433 0.4643 0.5230 0.0355 0.3175 2592 0.7752 0.5212 0.0881 0.2729 0.0504 0.3818 2594 0.6856 0.4905 0.8129 0.6041 0.5226 0.7738 2610 0.0753 0.2002 0.6346 0.5695 0.1788 0.5847 2624 0.1982 0.2712 0.6000 0.5693 0.0771 0.4764 2625 0.4206 0.3872 0.5346 0.5566 0.1614 0.5562 2649 0.0059 0.0536 0.0562 0.2376 0.3034 0.6748 2652 0.5148 0.4340 0.2860 0.4447 0.0938 0.5048 2669 0.0280 0.1320 0.1059 0.2848 0.5070 0.7596 3453 0.6988 0.4913 0.2050 0.3759 0.3697 0.7146 3454 0.9582 0.5712 0.1073 0.2848 0.1181 0.5242 3455 0.8397 0.5354 0.0643 0.2508 0.0428 0.3453 3456 0.4812 0.4241 0.1233 0.3029 0.0306 0.2992 3458 0.0267 0.1275 0.0016 0.0422 0.2238 0.6097 3461 0.1794 0.2616 0.7325 0.5835 0.3086 0.6785 3462 0.7023 0.4913 0.0904 0.2777 0.0425 0.3453 3464 0.0047 0.0517 0.0841 0.2676 0.1882 0.5850 3466 0.2729 0.3117 0.5765 0.5622 0.1056 0.5131 3471 0.0565 0.1757 0.0212 0.1420 0.6393 0.8251 3472 0.7507 0.5113 0.2731 0.4282 0.4303 0.7283 3473 0.0162 0.0939 0.3477 0.4761 0.1119 0.5146 3474 0.0016 0.0282 0.0172 0.1295 0.3091 0.6785 3478 0.7286 0.5004 0.3977 0.4995 0.6140 0.8107 3479 0.8920 0.5563 0.0659 0.2508 0.0503 0.3818 3480 0.5085 0.4333 0.8352 0.6041 0.6488 0.8251 3485 0.3407 0.3473 0.8628 0.6114 0.2632 0.6364 3495 0.1476 0.2433 0.9375 0.6284 0.1285 0.5309 3498 0.3963 0.3754 0.8121 0.6041 0.5384 0.7770 3505 0.4588 0.4111 0.1039 0.2848 0.3543 0.7016 3507 0.3431 0.3473 0.7293 0.5835 0.5422 0.7770 3508 0.1538 0.2443 0.0806 0.2676 0.7243 0.8380 3513 0.1333 0.2311 0.1841 0.3746 0.8522 0.8610 3514 0.1222 0.2311 0.2607 0.4221 0.6530 0.8251 3516 0.5175 0.4340 0.9399 0.6284 0.4708 0.7431 3522 0.1013 0.2257 0.3453 0.4761 0.4614 0.7415 3528 0.4692 0.4181 0.4296 0.5036 0.1383 0.5562 3530 0.8917 0.5563 0.6426 0.5695 0.7424 0.8380 3533 0.7342 0.5011 0.0145 0.1190 0.0303 0.2992 3534 0.7285 0.5004 0.0019 0.0462 0.0042 0.0925 3535 0.1140 0.2311 0.2034 0.3759 0.7393 0.8380 3537 0.7091 0.4932 0.6344 0.5695 0.9177 0.8691 3541 0.2248 0.2893 0.1794 0.3736 0.8911 0.8658 3542 0.8063 0.5274 0.2310 0.3905 0.1534 0.5562 3547 0.3238 0.3458 0.0066 0.0842 0.0572 0.4034 3548 0.0742 0.2002 0.0070 0.0854 0.2742 0.6411 3549 0.0183 0.0987 0.4057 0.5016 0.1008 0.5102 3550 0.0631 0.1838 0.8948 0.6233 0.0484 0.3787 3552 0.1138 0.2311 0.1875 0.3753 0.0068 0.1213 3563 0.0294 0.1325 0.6125 0.5695 0.0820 0.4886 3565 0.5592 0.4491 0.8370 0.6041 0.7038 0.8380 3566 0.1183 0.2311 0.7143 0.5835 0.0588 0.4034 3567 0.1998 0.2715 0.8892 0.6233 0.2500 0.6228 3568 0.6043 0.4610 0.0084 0.0919 0.0265 0.2876 3572 0.1181 0.2311 0.2684 0.4245 0.6263 0.8182 3573 0.3454 0.3473 0.4205 0.5036 0.0888 0.4997 3576 0.6347 0.4691 0.9016 0.6245 0.7246 0.8380 3577 0.1196 0.2311 0.0177 0.1305 0.3492 0.7016 3578 0.7635 0.5185 0.3616 0.4766 0.2298 0.6153 3579 0.9242 0.5621 0.6417 0.5695 0.5758 0.7827 3580 0.3075 0.3362 0.7120 0.5835 0.5088 0.7600 3585 0.0947 0.2167 0.6588 0.5739 0.2063 0.5984 3587 0.0564 0.1757 0.0088 0.0919 0.3910 0.7252 3588 0.2554 0.2999 0.8628 0.6114 0.1933 0.5864 3589 0.3985 0.3757 0.8302 0.6041 0.5259 0.7740 3598 0.3169 0.3414 0.0399 0.2100 0.2552 0.6290 3599 0.1514 0.2443 0.4277 0.5036 0.5029 0.7580 3602 0.1067 0.2298 0.4316 0.5036 0.3860 0.7252 3603 0.5341 0.4424 0.8982 0.6233 0.4548 0.7415 3607 0.0136 0.0833 0.0083 0.0919 0.8241 0.8538 3608 0.0293 0.1325 0.0657 0.2508 0.6931 0.8380 3624 0.9279 0.5621 0.9314 0.6284 0.9965 0.8795 3627 0.1279 0.2311 0.8037 0.6041 0.1964 0.5917 3628 0.9903 0.5795 0.7214 0.5835 0.7305 0.8380 3636 0.3080 0.3362 0.8352 0.6041 0.4134 0.7283 3641 0.0142 0.0854 0.5193 0.5467 0.0033 0.0776 3643 0.0023 0.0325 0.1619 0.3616 0.0001 0.0031 3644 0.0087 0.0665 0.6521 0.5695 0.0031 0.0771 3648 0.3678 0.3597 0.2309 0.3905 0.7567 0.8380 3649 0.0801 0.2064 0.9139 0.6254 0.0650 0.4168 3650 0.7836 0.5240 0.4937 0.5353 0.6798 0.8357 3651 0.0112 0.0738 0.2116 0.3803 0.0006 0.0192 3653 0.0149 0.0877 0.0261 0.1631 0.7961 0.8458 3654 0.8166 0.5274 0.0201 0.1413 0.0121 0.1630 3656 0.5640 0.4500 0.6367 0.5695 0.9158 0.8691 3658 0.2269 0.2893 0.1717 0.3733 0.8661 0.8610 3662 0.2810 0.3162 0.2413 0.4006 0.9217 0.8691 3663 0.1609 0.2443 0.3205 0.4661 0.6665 0.8314 3664 0.0570 0.1757 0.0158 0.1236 0.5441 0.7770 3667 0.1378 0.2318 0.2031 0.3759 0.8206 0.8519 3668 0.4944 0.4305 0.5523 0.5606 0.9280 0.8691 3670 0.0837 0.2140 0.1951 0.3759 0.6368 0.8251 3694 0.0023 0.0325 0.5766 0.5622 0.0085 0.1331 3698 0.3311 0.3473 0.3420 0.4761 0.0629 0.4168 3714 0.0104 0.0719 0.0090 0.0919 0.9463 0.8691 3715 0.0704 0.1968 0.3434 0.4761 0.3581 0.7028 3718 0.1859 0.2619 0.4513 0.5197 0.5542 0.7770 3727 0.5952 0.4584 0.0421 0.2154 0.1184 0.5242 3728 0.5890 0.4576 0.8945 0.6233 0.5022 0.7580 3751 0.2320 0.2930 0.0020 0.0462 0.0312 0.2992 3754 0.4775 0.4222 0.1407 0.3366 0.0353 0.3175 3757 0.0036 0.0444 1.0000 0.6499 0.0036 0.0827 3771 0.8174 0.5274 0.7150 0.5835 0.5524 0.7770 3978 0.3288 0.3473 0.2446 0.4041 0.8450 0.8610 4000 0.0480 0.1705 0.0082 0.0919 0.4183 0.7283 4079 0.1101 0.2298 0.0023 0.0479 0.0850 0.4957 4108 0.0005 0.0121 0.2899 0.4489 0.0059 0.1143 4131 0.6703 0.4860 0.6994 0.5811 0.4197 0.7283 4144 0.0446 0.1640 0.3567 0.4766 0.2443 0.6224 4221 0.0001 0.0019 0.0450 0.2206 0.0082 0.1319 4246 0.0000 0.0015 0.0216 0.1420 0.0110 0.1537 4274 0.0052 0.0517 0.3017 0.4575 0.0004 0.0158 4363 0.0892 0.2167 0.4952 0.5353 0.2879 0.6507 4756 0.2067 0.2747 0.7881 0.6001 0.3141 0.6797 4832 0.0326 0.1393 0.0580 0.2403 0.7786 0.8418 4835 0.0010 0.0234 0.4269 0.5036 0.0064 0.1195 4899 0.0440 0.1640 0.1216 0.3029 0.0012 0.0380 4926 1.0000 0.5814 0.0051 0.0717 0.0051 0.1021 4930 0.0000 0.0007 0.5097 0.5413 0.0001 0.0026 4971 0.0559 0.1757 0.0703 0.2551 0.9080 0.8681

Example 9: Table 5 for Examples 1-3, Provided as Parts Tables 5A Though 5F

Tables 5A through 5F (collectively “Table 5”) relate to the top 22 maternal serum and fetal brain metabolites downregulated in GF and ABX relative to SPF and Sp. The cells can be classified from the given data based on p<0.05 or 0.05<p<0.10, as well as based on the mean values being significantly higher or not for each comparison. In addition, the biochemicals found unpregulated in SPF and Sp compared to ABX and GF in both serum and brain can be extracted from the provided data (imidazole propionate; N,N,N-trimethyl-5-aminovalerate; 3-indoxyl sulfate; trimethylamine N-oxide; biotin; hippurate; stachydrine; pyrraline).

Tables 5A through 5C provide data for maternal serum, whereas Tables 5D through 5F provide data for fetal brain. Tables 5A and 5D provide “fold of change,” and the remaining sub-tables of Table 5 provide the ANOVA contrasts.

TABLE 5A Biochemical Name Sp/SPF ABX/SPF GF/SPF Sp/ABX GF/ABX GF/Sp alpha-ketoglutaramate* 1.37 0.61 0.49 2.23 0.81 0.36 imidazole propionate 2.40 0.35 0.22 6.90 0.62 0.09 N,N,N-trimethyl-5-aminovalerate 2.37 0.13 0.19 18.06 1.45 0.08 indolepropionate 0.70 0.01 0.00 54.72 0.24 0.00 3-indoxyl sulfate 0.52 0.43 0.00 1.20 0.00 0.00 trimethylamine N-oxide 0.36 0.16 0.02 2.33 0.10 0.04 beta-muricholate 6.42 0.01 0.01 676.89 1.27 0.00 alpha-muricholate 8.04 0.04 0.04 221.52 1.00 0.00 deoxycholate 1.69 0.01 0.02 115.92 1.05 0.01 taurodeoxycholate 0.07 0.00 0.00 21.07 1.00 0.05 ursodeoxycholate 3.73 0.02 0.02 201.13 1.00 0.00 taurohyodeoxycholic acid 0.16 0.01 0.00 17.84 0.47 0.03 7-ketodeoxycholate 3.31 0.00 0.00 717.10 1.00 0.00 biotin 1.50 0.44 0.44 3.41 0.99 0.29 hippurate 0.47 0.02 0.02 23.99 0.78 0.03 p-cresol sulfate 0.59 0.02 0.02 30.11 1.04 0.03 phenylpropionylglycine 0.60 0.01 0.01 72.57 1.00 0.01 3-(3-hydroxyphenyl)propionate sulfate 0.07 0.01 0.01 7.24 1.00 0.14 2-(4-hydroxyphenyl)propionate 0.99 0.06 0.06 16.71 1.00 0.06 3-phenylpropionate (hydrocinnamate) 1.72 0.07 0.12 25.21 1.72 0.07 stachydrine 0.92 0.63 0.59 1.45 0.94 0.65 pyrraline 1.03 0.59 0.44 1.73 0.74 0.43

TABLE 5B Sp/SPF ABX/SPF GF/SPF Biochemical Name p-value q-value p-value q-value p-value q-value alpha-ketoglutaramate* 0.1410 0.7037 0.0422 0.2485 0.0048 0.0281 imidazole propionate 0.0014 0.0451 0.0001 0.0021 0.0000 0.0000 N,N,N-trimethyl-5-aminovalerate 0.0001 0.0038 0.0000 0.0000 0.0000 0.0000 indolepropionate 0.4484 0.9734 0.0000 0.0000 0.0000 0.0000 3-indoxyl sulfate 0.1140 0.6811 0.0350 0.2157 0.0000 0.0000 trimethylamine N-oxide 0.0259 0.3001 0.0001 0.0010 0.0000 0.0000 beta-muricholate 0.0245 0.2975 0.0000 0.0000 0.0000 0.0000 alpha-muricholate 0.0457 0.3999 0.0010 0.0117 0.0010 0.0068 deoxycholate 0.3135 0.9011 0.0000 0.0000 0.0000 0.0000 taurodeoxycholate 0.0263 0.3001 0.0000 0.0000 0.0000 0.0000 ursodeoxycholate 0.0259 0.3001 0.0000 0.0000 0.0000 0.0000 taurohyodeoxycholic acid 0.2745 0.8757 0.0000 0.0007 0.0000 0.0000 7-ketodeoxycholate 0.1494 0.7164 0.0000 0.0000 0.0000 0.0000 biotin 0.0342 0.3258 0.0001 0.0011 0.0001 0.0007 hippurate 0.1238 0.7020 0.0000 0.0000 0.0000 0.0000 p-cresol sulfate 0.3343 0.9196 0.0000 0.0000 0.0000 0.0000 phenylpropionylglycine 0.2613 0.8757 0.0000 0.0000 0.0000 0.0000 3-(3-hydroxyphenyl)propionate sulfate 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 2-(4-hydroxyphenyl)propionate 0.3603 0.9196 0.0000 0.0000 0.0000 0.0000 3-phenylpropionate (hydrocinnamate) 0.8014 1.0000 0.0000 0.0000 0.0000 0.0000 stachydrine 0.6362 0.9975 0.0247 0.1690 0.0090 0.0468 pyrraline 0.8498 1.0000 0.0673 0.3424 0.0002 0.0014

TABLE 5C Sp/ABX GF/ABX GF/Sp Biochemical Name p-value q-value p-value q-value p-value q-value alpha-ketoglutaramate* 0.0009 0.0093 0.3789 0.9355 0.0001 0.0005 imidazole propionate 0.0000 0.0000 0.0451 0.4249 0.0000 0.0000 N,N,N-trimethyl-5-aminovalerate 0.0000 0.0000 0.0781 0.5552 0.0000 0.0000 indolepropionate 0.0000 0.0000 0.3756 0.9355 0.0000 0.0000 3-indoxyl sulfate 0.5737 0.6872 0.0000 0.0000 0.0000 0.0000 trimethylamine N-oxide 0.0369 0.1821 0.0000 0.0000 0.0000 0.0000 beta-muricholate 0.0000 0.0000 0.6005 1.0000 0.0000 0.0000 alpha-muricholate 0.0000 0.0000 1.0000 1.0000 0.0000 0.0000 deoxycholate 0.0000 0.0000 0.8840 1.0000 0.0000 0.0000 taurodeoxycholate 0.0000 0.0003 1.0000 1.0000 0.0000 0.0002 ursodeoxycholate 0.0000 0.0000 1.0000 1.0000 0.0000 0.0000 taurohyodeoxycholic acid 0.0011 0.0112 0.3075 0.9193 0.0000 0.0005 7-ketodeoxycholate 0.0000 0.0000 1.0000 1.0000 0.0000 0.0000 biotin 0.0000 0.0000 0.9763 1.0000 0.0000 0.0000 hippurate 0.0000 0.0000 0.4182 0.9628 0.0000 0.0000 p-cresol sulfate 0.0000 0.0000 0.9657 1.0000 0.0000 0.0000 phenylpropionylglycine 0.0000 0.0000 1.0000 1.0000 0.0000 0.0000 3-(3-hydroxyphenyl)propionate sulfate 0.0000 0.0000 1.0000 1.0000 0.0000 0.0000 2-(4-hydroxyphenyl)propionate 0.0000 0.0000 1.0000 1.0000 0.0000 0.0000 3-phenylpropionate (hydrocinnamate) 0.0000 0.0000 0.2181 0.8254 0.0000 0.0000 stachydrine 0.0706 0.2607 0.6835 1.0000 0.0288 0.0948 pyrraline 0.0448 0.2021 0.0280 0.3460 0.0001 0.0008

TABLE 5D Biochemical Name Sp/SPF ABX/SPF GF/SPF Sp/ABX GF/ABX GF/Sp glutamine 0.86 0.70 0.67 1.23 0.96 0.78 alpha-ketoglutaramate* 1.87 0.77 0.63 2.42 0.81 0.34 pyroglutamine* 0.97 0.73 0.51 1.34 0.70 0.52 imidazole propionate 1.60 0.33 0.33 4.81 0.99 0.20 anserine 0.82 0.40 0.42 2.04 1.04 0.51 N2-acetyllysine 0.88 0.49 0.58 1.80 1.18 0.66 N6-methyllysine 0.86 0.64 0.57 1.35 0.89 0.66 N,N,N-trimethyl-5-aminovalerate 1.79 0.09 0.16 19.45 1.71 0.09 3-indoxyl sulfate 0.56 0.31 0.08 1.79 0.25 0.14 3-sulfo-L-alanine 0.92 0.37 0.40 2.49 1.08 0.43 phenylacetylglycine 0.80 0.45 0.45 1.79 1.00 0.56 arachidoylcarnitine (C20)* 1.03 0.75 0.83 1.36 1.10 0.80 trimethylamine N-oxide 0.38 0.08 0.04 4.61 0.48 0.10 sphingomyelin (d18:1/20:0, d16:1/22:0)* 0.84 0.68 0.69 1.24 1.02 0.83 trigonelline (N′-methylnicotinate) 0.90 0.58 0.55 1.55 0.95 0.61 pantothenate 0.98 0.80 0.72 1.22 0.90 0.74 biotin 0.81 0.37 0.44 2.17 1.17 0.54 hippurate 0.42 0.24 0.24 1.72 1.00 0.58 homostachydrine* 0.72 0.38 0.46 1.91 1.24 0.65 stachydrine 0.77 0.44 0.57 1.75 1.28 0.73 pyrraline 0.98 0.46 0.40 2.14 0.87 0.41 O-sulfo-L-tyrosine 0.95 0.47 0.58 2.01 1.22 0.61

TABLE 5E Sp/SPF ABX/SPF GF/SPF Biochemical Name p-value q-value p-value q-value p-value q-value glutamine 0.0903 0.0627 0.0010 0.0037 0.0003 0.0015 alpha-ketoglutaramate* 0.0006 0.0059 0.1065 0.0509 0.0061 0.0089 pyroglutamine* 0.9870 0.3040 0.0347 0.0252 0.0001 0.0006 imidazole propionate 0.0121 0.0243 0.0000 0.0001 0.0000 0.0001 anserine 0.5221 0.1970 0.0042 0.0068 0.0025 0.0052 N2-acetyllysine 0.3313 0.1431 0.0010 0.0038 0.0049 0.0076 N6-methyllysine 0.1851 0.0975 0.0010 0.0037 0.0001 0.0006 N,N,N-trimethyl-5-aminovalerate 0.0019 0.0111 0.0000 0.0000 0.0000 0.0000 3-indoxyl sulfate 0.0326 0.0377 0.0000 0.0005 0.0000 0.0000 3-sulfo-L-alanine 0.5338 0.2006 0.0000 0.0000 0.0000 0.0001 phenylacetylglycine 0.5433 0.2032 0.0079 0.0096 0.0079 0.0107 arachidoylcarnitine (C20)* 0.7317 0.2468 0.0046 0.0069 0.0445 0.0332 trimethylamine N-oxide 0.0018 0.0110 0.0000 0.0000 0.0000 0.0000 sphingomyelin (d18:1/20:0, d16:1/22:0)* 0.1795 0.0960 0.0045 0.0069 0.0048 0.0076 trigonelline (N′-methylnicotinate) 0.4532 0.1780 0.0025 0.0055 0.0005 0.0022 pantothenate 0.6854 0.2376 0.0044 0.0069 0.0002 0.0014 biotin 0.4864 0.1893 0.0004 0.0025 0.0017 0.0038 hippurate 0.0000 0.0008 0.0000 0.0000 0.0000 0.0000 homostachydrine* 0.0336 0.0377 0.0000 0.0001 0.0000 0.0004 stachydrine 0.0223 0.0321 0.0000 0.0000 0.0000 0.0003 pyrraline 0.8733 0.2799 0.0036 0.0064 0.0003 0.0016 O-sulfo-L-tyrosine 0.7093 0.2411 0.0004 0.0025 0.0027 0.0053

TABLE 5F Sp/ABX GF/ABX GF/Sp Biochemical Name p-value q-value p-value q-value p-value q-value glutamine 0.0500 0.1713 0.5935 0.5688 0.0161 0.2066 alpha-ketoglutaramate* 0.0000 0.0007 0.1850 0.3746 0.0000 0.0001 pyroglutamine* 0.0359 0.1496 0.0114 0.1024 0.0001 0.0026 imidazole propionate 0.0000 0.0000 0.9507 0.6291 0.0000 0.0000 anserine 0.0181 0.0987 0.8231 0.6041 0.0111 0.1537 N2-acetyllysine 0.0100 0.0718 0.5070 0.5413 0.0423 0.3453 N6-methyllysine 0.0217 0.1120 0.2581 0.4219 0.0016 0.0466 N,N,N-trimethyl-5-aminovalerate 0.0000 0.0000 0.0059 0.0770 0.0000 0.0000 3-indoxyl sulfate 0.0078 0.0610 0.0000 0.0068 0.0000 0.0000 3-sulfo-L-alanine 0.0000 0.0004 0.5749 0.5622 0.0000 0.0009 phenylacetylglycine 0.0303 0.1333 1.0000 0.6499 0.0303 0.2992 arachidoylcarnitine (C20)* 0.0021 0.0325 0.3098 0.4633 0.0216 0.2516 trimethylamine N-oxide 0.0000 0.0008 0.0089 0.0919 0.0000 0.0000 sphingomyelin (d18:1/20:0, d16:1/22:0)* 0.0863 0.2155 0.9790 0.6396 0.0907 0.5044 trigonelline (N′-methylnicotinate) 0.0142 0.0854 0.5193 0.5467 0.0033 0.0776 pantothenate 0.0112 0.0738 0.2116 0.3803 0.0006 0.0192 biotin 0.0023 0.0325 0.5766 0.5622 0.0085 0.1331 hippurate 0.0036 0.0444 1.0000 0.6499 0.0036 0.0827 homostachydrine* 0.0005 0.0121 0.2899 0.4489 0.0059 0.1143 stachydrine 0.0001 0.0019 0.0450 0.2206 0.0082 0.1319 pyrraline 0.0052 0.0517 0.3017 0.4575 0.0004 0.0158 O-sulfo-L-tyrosine 0.0010 0.0234 0.4269 0.5036 0.0064 0.1195

INCORPORATION BY REFERENCE

All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

EQUIVALENTS

While specific embodiments of the subject invention have been discussed, the above specification is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art upon review of this specification and the claims below. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations. 

1. A method of promoting healthy neural development in an unborn baby, the method comprising administering to a maternal subject gestating the unborn baby a composition comprising trimethylamine N-oxide (TMAO), 5-aminovalerate (5-AV), imidazole propionate (IP), hippurate (HIP), or a combination thereof.
 2. The method of claim 1, wherein the composition comprises 5-AV and IP.
 3. The method of claim 1, wherein the composition comprises TMAO.
 4. The method of claim 1, wherein said healthy neural development comprises healthy thalamocortical axon growth.
 5. The method of claim 1, wherein said healthy neural development comprises healthy netrin-G1a+thalamocortical axogenesis.
 6. The method of claim 1, wherein the maternal subject and the unborn baby are mammals.
 7. The method of claim 1, wherein the maternal subject and the unborn baby are humans.
 8. The method of claim 7, wherein the method comprises administering the composition at least once during the first trimester of the gestating maternal subject's gestation period.
 9. The method of claim 8, wherein the method comprises administering the composition at least once during a period that runs from the start of the third week after conception to the end of the eighth week after conception.
 10. The method of claim 8, wherein the method comprises administering the composition at least once during a period that runs from the 17^(th) day post conception (dpc) to the 52^(nd) dpc.
 11. The method of claim 7, wherein the method comprises administering the composition at least once during the second trimester of the gestating maternal subject's gestation period.
 12. The method of claim 7, wherein the method comprises administering the composition at least once during the third trimester of the gestating maternal subject's gestation period.
 13. The method of claim 1, wherein the unborn baby is an offspring of the maternal subject.
 14. A method of conditioning a female subject for fostering healthy neural development in offspring, the method comprising administering to the female subject a composition comprising trimethylamine N-oxide (TMAO), 5-aminovalerate (5-AV), imidazole propionate (IP), hippurate (HIP), or a combination thereof, wherein the composition is administered at least once during a period that runs from the first day of an expected-but-missed menstruation to a day that is two months after said first day.
 15. The method of claim 14, wherein the composition is administered at least once during a period that runs from the second day of the expected-but-missed menstruation to a day that is 37 days after said second day.
 16. The method of claim 14, wherein said healthy neural development comprises healthy tactile sensory development.
 17. A method of promoting healthy neural development in an unborn baby, the method comprising administering to a maternal subject gestating the unborn baby a bacterial composition comprising bacteria of the order Clostridiales.
 18. The method of claim 17, wherein said bacteria of the order Clostridiales comprise bacteria of the family Lachnospiraceae, family Ruminococcaceae, family Clostridiaceae, or a combination thereof.
 19. The method of claim 17, wherein said bacteria of the order Clostridiales comprise bacteria of the genus Clostridium, genus Dehalobacterium, genus Ruminococcus, genus Coprococcus, genus Dorea, genus Oscillospira, or a combination thereof.
 20. The method of claim 17, wherein said bacteria of the order Clostridiales are spore-forming bacteria.
 21. The method of claim 17, wherein the method comprises administering the bacterial composition at least once during the first trimester of the gestating maternal subject's gestation period.
 22. The method of claim 17, wherein the method further comprises administering the bacterial composition at least once during the two-month period before said gestation period starts.
 23. The method of claim 17, wherein the method further comprises administering to the maternal subject a composition comprising trimethylamine N-oxide (TMAO), 5-aminovalerate (5-AV), imidazole propionate (IP), hippurate (HIP), or a combination thereof.
 24. A method of conditioning a female subject for bringing about offspring with healthy neural development, the method comprising administering to the female subject a bacterial composition comprising spore-forming bacteria of the order Clostridiales, wherein the bacterial composition is administered at least once during a two-month period that ends with the day of an expected conception for the female subject.
 25. A method of reducing adverse effects of antibiotic treatment on an unborn baby in a pregnant subject, the method comprising administering to the pregnant subject, conjointly with the antibiotic treatment, a composition comprising trimethylamine N-oxide (TMAO), 5-aminovalerate (5-AV), imidazole propionate (IP), hippurate (HIP), or a combination thereof.
 26. A method of selecting a female subject for conditioning to foster healthy neural development in offspring, the method comprising determining that a compound selected from 2-(4-hydroxyphenyl)propionate; 3-(3-hydroxyphenyl)propionate sulfate; 3-indoxyl sulfate; 3-phenylpropionate (hydrocinnamate); 7-ketodeoxycholate; alpha-ketoglutaramate; alpha-muricholate; beta-muricholate; biotin; deoxycholate; hippurate; imidazole propionate; indolepropionate; N,N,N-trimethyl-5-aminovalerate; p-cresol sulfate; phenylpropionylglycine; pyrraline; stachydrine; taurodeoxycholate; taurohyodeoxycholic acid; trimethylamine N-oxide; ursodeoxycholate; and a combination thereof has a level in a serum sample from the female subject that is at most 10%, 20%, 30%, 40%, 50%, 60%, or 70% of its level in a control serum sample representative of a healthy female subject; bacteria of the order Clostridiales have a total level in a fecal sample from the female subject that is at most 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 14%, 16%, 18%, or 20% of their total level in a control fecal sample representative of a healthy female subject; or a combination thereof; and selecting the female subject for conditioning to foster healthy neural development in offspring.
 27. The method of claim 26, further comprising administering to the female subject a composition comprising trimethylamine N-oxide (TMAO), 5-aminovalerate (5-AV), imidazole propionate (IP), hippurate (HIP), or a combination thereof; a bacterial composition comprising spore-forming bacteria of the order Clostridiales; or a combination thereof.
 28. The method of claim 26, wherein the compound is selected from 3-indoxyl sulfate; biotin; hippurate; imidazole propionate; N,N,N-trimethyl-5-aminovalerate; pyrraline; stachydrine; trimethylamine N-oxide; and a combination thereof; and the bacteria of the order Clostridiales are selected from bacteria of the genus Clostridium, genus Dehalobacterium, genus Ruminococcus, genus Coprococcus, genus Dorea, genus Oscillospira, and a combination thereof.
 29. The method of claim 26, further comprising using liquid chromatography-mass spectrometry to determine a level for the compound.
 30. The method of claim 26, further comprising using 16S rDNA sequencing to determine a total level for the bacteria. 